Polypeptides Having Cellulolytic Enhancing Activity and Polynucleotides Encoding Same

ABSTRACT

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/979,223 filed Feb. 23, 2012, which is a 35 U.S.C. §371 nationalapplication of PCT/CN2012/071525 filed Feb. 23, 2012, which claimspriority or the benefit under 35 U.S.C. §119 of PCT Application No.PCT/CN2011/071208 filed Feb. 23, 2011 and U.S. Provisional ApplicationNo. 61/471,423 filed Apr. 4, 2011, the contents of which are fullyincorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under CooperativeAgreement DE-FC36-08GO18080 awarded by the Department of Energy. Thegovernment has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to polypeptides having cellulolyticenhancing activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods of producing and using the polypeptides.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4 bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose. Once the cellulose is converted toglucose, the glucose is easily fermented by yeast into ethanol.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin.

WO 2005/074647, WO 2008/148131, WO 2011/035027 disclose isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Thielavia terrestris. WO 2005/074656 and WO2010/065830 disclose isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascusaurantiacus. WO 2007/089290 discloses an isolated GH61 polypeptidehaving cellulolytic enhancing activity and the polynucleotide thereoffrom Trichoderma reesei. WO 2009/085935, WO 2009/085859, WO 2009/085864,and WO 2009/085868 disclose isolated GH61 polypeptides havingcellulolytic enhancing activity and the polynucleotides thereof fromMyceliophthora thermophila. WO 2010/138754 discloses isolated GH61polypeptides having cellulolytic enhancing activity and thepolynucleotides thereof from Aspergillus fumigatus. WO 2011/005867discloses isolated GH61 polypeptides having cellulolytic enhancingactivity and the polynucleotides thereof from Penicillium pinophilum. WO2011/039319 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascus sp.WO 2011/041397 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Penicillium sp.WO 2011/041504 discloses isolated GH61 polypeptides having cellulolyticenhancing activity and the polynucleotides thereof from Thermoascuscrustaceous. WO 2008/151043 discloses methods of increasing the activityof a GH61 polypeptide having cellulolytic enhancing activity by adding asoluble activating divalent metal cation to a composition comprising thepolypeptide.

There is a need in the art for new enzymes to increase efficiency and toprovide cost-effective enzyme solutions for saccharification ofcellulosic material. The present invention provides GH61 polypeptideshaving cellulolytic enhancing activity and polynucleotides encoding thepolypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellulolytic enhancing activity selected from the group consisting of:

(a) a polypeptide having at least 70% sequence identity to the maturepolypeptide of SEQ ID NO: 2, or at least 60% sequence identity to themature polypeptide of SEQ ID NO: 4, or at least 70% sequence identity tothe mature polypeptide of SEQ ID NO: 6;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast medium stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) thecDNA sequence contained in the mature polypeptide coding sequence of SEQID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) a full-lengthcomplementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 70%sequence identity to the mature polypeptide of SEQ ID NO: 1, or at least60% sequence identity to the mature polypeptide of SEQ ID NO: 3, or atleast 70% sequence identity to the mature polypeptide of SEQ ID NO: 5;

(d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4,or SEQ ID NO: 6 comprising a substitution, deletion, and/or insertion ofone or more (e.g., several) amino acids; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to isolated polynucleotides encodingpolypeptides having cellulolytic enhancing activity, selected from thegroup consisting of:

(a) a polynucleotide encoding a polypeptide having at least 70% sequenceidentity to the mature polypeptide of SEQ ID NO: 2, or at least 60%sequence identity to the mature polypeptide of SEQ ID NO: 4, or at least70% sequence identity to the mature polypeptide of SEQ ID NO: 6;

(b) a polynucleotide that hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) the cDNA sequence contained inthe mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, orSEQ ID NO: 5, or (iii) a full-length complementary strand of (i) or(ii);

(c) a polynucleotide having at least 70% sequence identity to the maturepolypeptide of SEQ ID NO: 1, or at least 60% sequence identity to themature polypeptide of SEQ ID NO: 3, or at least 70% sequence identity tothe mature polypeptide of SEQ ID NO: 5;

(d) a polynucleotide encoding a variant of SEQ ID NO: 2, SEQ ID NO: 4,or SEQ ID NO: 6 comprising a substitution, deletion, and/or insertion ofone or more (e.g., several) amino acids of the mature polypeptide; and

(e) a fragment of the polynucleotide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, recombinant host cells comprising thepolynucleotides, and methods of producing the polypeptides havingcellulolytic enhancing activity.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellulolytic enhancing activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. The presentalso relates to such a double-stranded RNA (dsRNA) molecule, whereinoptionally the dsRNA is an siRNA or an miRNA molecule.

The present invention also relates to compositions comprising thepolypeptide of the present invention.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention. In apreferred aspect, the method further comprises recovering the degradedor converted cellulosic material.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention. In a preferred aspect, the fermenting of the cellulosicmaterial produces a fermentation product. In one aspect, the methodfurther comprises recovering the fermentation product from thefermentation.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding a polypeptide having cellulolytic enhancingactivity.

The present invention also relates to methods of producing a polypeptidehaving cellulolytic enhancing activity, comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide having cellulolytic enhancing activity under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

The present invention further relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to22 of SEQ ID NO: 2, amino acids 1 to 21 of SEQ ID NO: 4, or amino acids1 to 22 of SEQ ID NO: 6; to nucleic acid constructs, expression vectors,and recombinant host cells comprising the polynucleotides; and methodsof producing a protein.

The present invention further relates to a whole broth formulation orcell culture composition comprising the polypeptide of the presentinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thermomyces lanuginosus GH61(1)polypeptide having cellulolytic enhancing activity (SEQ ID NOs: 1 and 2,respectively).

FIG. 2 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thermomyces lanuginosus GH61(2)polypeptide having cellulolytic enhancing activity (SEQ ID NOs: 3 and 4,respectively).

FIG. 3 shows the genomic DNA sequence and the deduced amino acidsequence of a gene encoding a Thermomyces lanuginosus GH61(3)polypeptide having cellulolytic enhancing activity (SEQ ID NOs: 5 and 6,respectively).

FIG. 4 shows the alignment of SEQ ID NOs: 2 and 4.

FIG. 5 shows the alignment of SEQ ID NOs: 2 and 6.

FIG. 6 shows the alignment of SEQ ID NOs: 4 and 6.

DEFINITIONS

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 30° C., 50° C., 55° C., or 60°C., and pH, e.g., 5.0 or 5.5, compared to a control hydrolysis withequal total protein loading without cellulolytic enhancing activity(1-50 mg of cellulolytic protein/g of cellulose in PCS). In a preferredaspect, a mixture of CELLUCLAST® 1.5 L (Novozymes NS, Bagsværd, Denmark)in the presence of 2-3% of total protein weight Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus oryzae asdescribed in WO 2002/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

The polypeptides having cellulolytic enhancing activity have at least20%, preferably at least 40%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 70%, more preferablyat least 80%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 100% of the cellulolyticenhancing activity of the mature polypeptide of SEQ ID NO: 2, the maturepolypeptide of SEQ ID NO: 4 or the mature polypeptide of SEQ ID NO: 6.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, more preferably at least 1.05-fold, more preferably at least1.10-fold, more preferably at least 1.25-fold, more preferably at least1.5-fold, more preferably at least 2-fold, more preferably at least3-fold, more preferably at least 4-fold, more preferably at least5-fold, even more preferably at least 10-fold, and most preferably atleast 20-fold.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing ornon-reducing ends of the chain (Teeri, 1997, Crystalline cellulosedegradation: New insight into the function of cellobiohydrolases, Trendsin Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reeseicellobiohydrolases: why so efficient on crystalline cellulose?, Biochem.Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determinedaccording to the procedures described by Lever et al., 1972, Anal.Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149:152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288;and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the presentinvention, the Tomme et al. method can be used to determinecellobiohydrolase activity.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, Recent progress in the assays of xylanolytic enzymes, 2006,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek,1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctionalbeta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey, Biely, Poutanen, 1992,Interlaboratory testing of methods for assay of xylanase activity,Journal of Biotechnology 23(3): 257-270. Xylanase activity can also bedetermined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON®X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200mM sodium phosphate buffer pH 6 at 37° C. One unit of xylanase activityis defined as 1.0 μmole of azurine produced per minute at 37° C., pH 6from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom, D.and Shoham, Y. Microbial hemicellulases. Current Opinion InMicrobiology, 2003, 6(3): 219-228). Hemicellulases are key components inthe degradation of plant biomass. Examples of hemicellulases include,but are not limited to, an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase. The substrates of these enzymes, the hemicelluloses, are aheterogeneous group of branched and linear polysaccharides that arebound via hydrogen bonds to the cellulose microfibrils in the plant cellwall, crosslinking them into a robust network. Hemicelluloses are alsocovalently attached to lignin, forming together with cellulose a highlycomplex structure. The variable structure and organization ofhemicelluloses require the concerted action of many enzymes for itscomplete degradation. The catalytic modules of hemicellulases are eitherglycoside hydrolases (GHs) that hydrolyze glycosidic bonds, orcarbohydrate esterases (CEs), which hydrolyze ester linkages of acetateor ferulic acid side groups. These catalytic modules, based on homologyof their primary sequence, can be assigned into GH and CE families. Somefamilies, with an overall similar fold, can be further grouped intoclans, marked alphabetically (e.g., GH-A). A most informative andupdated classification of these and other carbohydrate active enzymes isavailable in the Carbohydrate-Active Enzymes (CAZy) database.Hemicellulolytic enzyme activities can be measured according to Ghoseand Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitabletemperature, e.g., 30° C., 50° C., 55° C., or 60° C., and pH, e.g., 5.0or 5.5.

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEX® HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, or neutralpretreatment.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Thepolypeptide of the present invention may be used in industrialapplications in the form of a fermentation broth product, that is, thepolypeptide of the present invention is a component of a fermentationbroth used as a product in industrial applications (e.g., ethanolproduction). The fermentation broth product will in addition to thepolypeptide of the present invention comprise additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. The fermentation broth may optionally be subjected to one ormore purification (including filtration) steps to remove or reduce onemore components of a fermentation process. Accordingly, an isolatedsubstance may be present in such a fermentation broth product.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 97%pure, more preferably at least 98% pure, even more preferably at least99% pure, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation. The polypeptides of the present invention are preferably ina substantially pure form, i.e., that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyor recombinantly associated. This can be accomplished, for example, bypreparing the polypeptide by well-known recombinant methods or byclassical purification methods.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 23 to 272 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 22 of SEQ ID NO: 2 are a signal peptide. Inanother aspect, the mature polypeptide is amino acids 22 to 327 of SEQID NO: 4 based on the SignalP program that predicts amino acids 1 to 21of SEQ ID NO: 4 are a signal peptide. In another aspect, the maturepolypeptide is amino acids 23 to 274 of SEQ ID NO: 6 based on theSignalP program that predicts amino acids 1 to 22 of SEQ ID NO: 6 are asignal peptide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellulolytic enhancing activity. In one aspect, the maturepolypeptide coding sequence is nucleotides 67 to 869 of SEQ ID NO: 1based on the SignalP program (Nielsen et al., 1997, supra) that predictsnucleotides 1 to 66 of SEQ ID NO: 1 encode a signal peptide. In anotheraspect, the mature polypeptide coding sequence is nucleotides 64 to 1036of SEQ ID NO: 3 based on the SignalP program that predicts nucleotides 1to 63 of SEQ ID NO: 3 encode a signal peptide. In one aspect, the maturepolypeptide coding sequence is nucleotides 67 to 878 of SEQ ID NO: 5based on the SignalP program that predicts nucleotides 1 to 66 of SEQ IDNO: 5 encode a signal peptide.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 3.0.0, 5.0.0 or later. The parametersused are gap open penalty of 10, gap extension penalty of 0.5, and theEBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the—nobrief option)is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 3.0.0, 5.0.0 or later. The parameters used are gap open penaltyof 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version ofNCBI NUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein having an E value (or expectancy score) of less than0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with the Thermomyces lanuginosus polypeptide having cellulolyticenhancing activity of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 or themature polypeptide thereof.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (e.g., several) amino acids deletedfrom the amino and/or carboxyl terminus of the mature polypeptide of SEQID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or a homologous sequencethereof; wherein the fragment has cellulolytic enhancing activity. In apreferred aspect, a fragment contains at least 190 amino acid residues,more preferably at least 200 amino acid residues, and most preferably atleast 210 amino acid residues, of the mature polypeptide of SEQ ID NO: 2or a homologous sequence thereof. In another preferred aspect, afragment contains at least 270 amino acid residues, more preferably atleast 290 amino acid residues, and most preferably at least 310 aminoacid residues, of the mature polypeptide of SEQ ID NO: 4 or a homologoussequence thereof. In another preferred aspect, a fragment contains atleast 190 amino acid residues, more preferably at least 200 amino acidresidues, and most preferably at least 210 amino acid residues, of themature polypeptide of SEQ ID NO: 6 or a homologous sequence thereof.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (e.g., several) nucleotides absent from the5′ and/or 3′ end of the mature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO: 5; or a homologous sequence thereof;wherein the subsequence encodes a polypeptide fragment havingcellulolytic enhancing activity. In a preferred aspect, a subsequencecontains at least 570 nucleotides, more preferably at least 600nucleotides, and most preferably at least 630 nucleotides of the maturepolypeptide coding sequence of SEQ ID NO: 1 or a homologous sequencethereof. In another preferred aspect, a subsequence contains at least810 nucleotides, more preferably at least 870 nucleotides, and mostpreferably at least 930 nucleotides of the mature polypeptide codingsequence of SEQ ID NO: 3 or a homologous sequence thereof. In anotherpreferred aspect, a subsequence contains at least 570 nucleotides, morepreferably at least 600 nucleotides, and most preferably at least 630nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 5 ora homologous sequence thereof.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99% pure, and even most preferably at least99.5% pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.These steps include the removal of intron sequences by a process calledsplicing. cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide comprising or consisting of the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or ahomologous sequence thereof; as well as genetic manipulation of the DNAencoding such a polypeptide. The modification can be a substitution, adeletion and/or an insertion of one or more (e.g., several) amino acidsas well as replacements of one or more (e.g., several) amino acid sidechains.

Variant: The term “variant” means a polypeptide having cellulolyticenhancing activity comprising an alteration, i.e., a substitution,insertion, and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having CellulolyticEnhancing Activity

In a first aspect, the present invention relates to isolatedpolypeptides comprising amino acid sequences having a degree of identityto the mature polypeptide of SEQ ID NO: 2 of preferably at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, or atleast 89%, more preferably at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, and most preferably at least 96%,at least 97%, at least 98%, at least 99%, or 100%, which havecellulolytic enhancing activity (hereinafter “homologous polypeptides”).In a preferred aspect, the homologous polypeptides comprise amino acidsequences that differ by preferably ten amino acids, by nine aminoacids, by eight amino acids, even preferably by seven amino acids, bysix amino acids, more preferably by five amino acids, by four aminoacids, even more preferably by three amino acids, most preferably by twoamino acids, and even most preferably by one amino acid from the maturepolypeptide of SEQ ID NO: 2.

In a preferable embodiment, the homologous polypeptide of SEQ ID NO: 2can be SEQ ID NO: 6. The mature polypeptide of SEQ ID NO: 2 has a degreeof identity to the mature polypeptide of SEQ ID NO: 6 of 84.9%.

In another first aspect, the present invention relates to isolatedpolypeptides comprising amino acid sequences having a degree of identityto the mature polypeptide of SEQ ID NO: 4 of preferably at least 60%, atleast 65%, at least 70%, at least 75%, even preferably at least 80%,more preferably at least 81%, at least 82%, at least 83%, at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,even more preferably at least 90%, at least 91%, at least 92%, at least93%, at least 94%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, at least 99%, or100%, which have cellulolytic enhancing activity (hereinafter“homologous polypeptides”). In a preferred aspect, the homologouspolypeptides comprise amino acid sequences that differ by preferably tenamino acids, by nine amino acids, by eight amino acids, even preferablyby seven amino acids, by six amino acids, more preferably by five aminoacids, by four amino acids, even more preferably by three amino acids,most preferably by two amino acids, and even most preferably by oneamino acid from the mature polypeptide of SEQ ID NO: 4.

In another first aspect, the present invention relates to isolatedpolypeptides comprising amino acid sequences having a degree of identityto the mature polypeptide of SEQ ID NO: 6 of preferably at least 70%, atleast 75%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, more preferably at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, most preferably at least 95%, and even mostpreferably at least 96%, at least 97%, at least 98%, at least 99%, or100%, which have cellulolytic enhancing activity (hereinafter“homologous polypeptides”). In a preferred aspect, the homologouspolypeptides comprise amino acid sequences that differ by ten aminoacids, by nine amino acids, by eight amino acids, even preferably byseven amino acids, by six amino acids, more preferably by five aminoacids, by four amino acids, even more preferably by three amino acids,most preferably by two amino acids, and even most preferably by oneamino acid from the mature polypeptide of SEQ ID NO: 6.

In a preferable embodiment, the homologous polypeptide of SEQ ID NO: 6can be SEQ ID NO: 2. The mature polypeptide of SEQ ID NO: 6 has a degreeof identity to SEQ ID NO: 2 of 84.9%.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof having cellulolytic enhancing activity. In a preferredaspect, the polypeptide comprises the amino acid sequence of SEQ ID NO:2. In another preferred aspect, the polypeptide comprises the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide comprises amino acids 23 to 272 of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 23 to 272 of SEQ ID NO: 2. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 2 or an allelic variant thereof; or a fragment thereof havingcellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, thepolypeptide consists of amino acids 23 to 272 of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 23 to 272 of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 4 or an allelic variant thereof; or afragment thereof having cellulolytic enhancing activity. In a preferredaspect, the polypeptide comprises the amino acid sequence of SEQ ID NO:4. In another preferred aspect, the polypeptide comprises the maturepolypeptide of SEQ ID NO: 4. In another preferred aspect, thepolypeptide comprises amino acids 22 to 327 of SEQ ID NO: 4, or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 22 to 327 of SEQ ID NO: 4. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 4 or an allelic variant thereof; or a fragment thereof havingcellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 4. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 4. In another preferred aspect, thepolypeptide consists of amino acids 22 to 327 of SEQ ID NO: 4 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 22 to 327 of SEQ ID NO: 4.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 6 or an allelic variant thereof; or afragment thereof having cellulolytic enhancing activity. In a preferredaspect, the polypeptide comprises the amino acid sequence of SEQ ID NO:6. In another preferred aspect, the polypeptide comprises the maturepolypeptide of SEQ ID NO: 6. In another preferred aspect, thepolypeptide comprises amino acids 23 to 274 of SEQ ID NO: 6, or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptidecomprises amino acids 23 to 274 of SEQ ID NO: 6. In another preferredaspect, the polypeptide consists of the amino acid sequence of SEQ IDNO: 6 or an allelic variant thereof; or a fragment thereof havingcellulolytic enhancing activity. In another preferred aspect, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 6. Inanother preferred aspect, the polypeptide consists of the maturepolypeptide of SEQ ID NO: 6. In another preferred aspect, thepolypeptide consists of amino acids 23 to 274 of SEQ ID NO: 6 or anallelic variant thereof; or a fragment thereof having cellulolyticenhancing activity. In another preferred aspect, the polypeptideconsists of amino acids 23 to 274 of SEQ ID NO: 6.

In a second aspect, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity that are encoded bypolynucleotides that hybridize under medium stringency conditions, ormedium-high stringency conditions, preferably high stringency conditionsand more preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a full-length complementary strand of (i) or (ii) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y.).

In another second aspect, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity that are encoded bypolynucleotides that hybridize under medium stringency conditions, ormedium-high stringency conditions, or preferably high stringencyconditions and more preferably very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 3, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 3, or (iii) a full-length complementary strand of (i) or (ii).

In another second aspect, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity that are encoded bypolynucleotides that hybridize under medium stringency conditions, ormedium-high stringency conditions, preferably high stringency conditionsand more preferably very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 5, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 5, or(iii) a full-length complementary strand of (i) or (ii). Thepolynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or asubsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 4, or SEQ ID NO: 6; or a fragment thereof; may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having cellulolytic enhancing activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic DNA or cDNA of a cell of interest, following standard

Southern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 15, preferably at least 25,more preferably at least 35, or at least 70 nucleotides in length.Preferably, the nucleic acid probe is at least 100 nucleotides inlength, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellulolytic enhancing activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridizes with SEQ ID NO: 1, SEQID NO: 3, or SEQ ID NO: 5, or a subsequence thereof, the carriermaterial is preferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; (ii) the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:5; (iii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; (iv) thefull-length complement thereof; or (v) a subsequence thereof; under verylow to very high stringency conditions. Molecules to which the nucleicacid probe hybridizes under these conditions can be detected using, forexample, X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1. In another preferred aspect, the nucleic acidprobe is nucleotides 67 to 869 of SEQ ID NO: 1. In another preferredaspect, the nucleic acid probe is a polynucleotide that encodes thepolypeptide of SEQ ID NO: 2, or a subsequence thereof. In anotherpreferred aspect, the nucleic acid probe is SEQ ID NO: 1. In anotheraspect, the nucleic acid probe is the polynucleotide contained inplasmid pGH61_(—)664 which is contained in E. coli CGMCC 4601, whereinthe polynucleotide thereof encodes a polypeptide having cellulolyticenhancing activity. In another preferred aspect, the nucleic acid probeis the mature polypeptide coding region contained in plasmidpGH61_(—)664 which is contained in E. coli CGMCC 4601.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 3. In another preferred aspect, the nucleic acidprobe is nucleotides 64 to 1036 of SEQ ID NO: 3. In another preferredaspect, the nucleic acid probe is a polynucleotide that encodes thepolypeptide of SEQ ID NO: 4, or a subsequence thereof. In anotherpreferred aspect, the nucleic acid probe is SEQ ID NO: 3. In anotheraspect, the nucleic acid probe is the polynucleotide contained inplasmid pGH61_(—)1590 which is contained in E. coli CGMCC 4602, whereinthe polynucleotide thereof encodes a polypeptide having cellulolyticenhancing activity. In another preferred aspect, the nucleic acid probeis the mature polypeptide coding region contained in plasmidpGH61_(—)1590 which is contained in E. coli CGMCC 4602.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 5. In another preferred aspect, the nucleic acidprobe is nucleotides 67 to 878 of SEQ ID NO: 5. In another preferredaspect, the nucleic acid probe is a polynucleotide that encodes thepolypeptide of SEQ ID NO: 6, or a subsequence thereof. In anotherpreferred aspect, the nucleic acid probe is SEQ ID NO: 5. In anotheraspect, the nucleic acid probe is the polynucleotide contained inplasmid pGH61_(—)4950 which is contained in E. coli CGMCC 4600, whereinthe polynucleotide thereof encodes a polypeptide having cellulolyticenhancing activity. In another preferred aspect, the nucleic acid probeis the mature polypeptide coding region contained in plasmidpGH61_(—)4950 which is contained in E. coli CGMCC 4600.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides comprising or consisting of nucleotide sequences thathave a degree of sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1 of at least 70%, e.g., at least 75%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, morepreferably at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, more preferably at least 95%, and most preferably at least96%, at least 97%, at least 98%, at least 99%, or 100%, which encode apolypeptide having cellulolytic enhancing activity.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides comprising or consisting of nucleotide sequences thathave a degree of sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 3 of preferably at least 60%, at least 65%, atleast 70%, at least 75%, even preferably at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, even more preferably at least90%, at least 91%, at least 92%, at least 93%, at least 94%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, at least 99%, or 100%, which encode a polypeptidehaving cellulolytic enhancing activity.

In another embodiment, the present invention relates to isolatedpolypeptides having cellulolytic enhancing activity encoded bypolynucleotides comprising or consisting of nucleotide sequences thathave a degree of identity to the mature polypeptide coding sequence ofSEQ ID NO: 5 of at least 70%, at least 75%, at least 80%, at least 81%,at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, more preferably at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, most preferably atleast 95%, and even most preferably at least 96%, at least 97%, at least98%, at least 99%, or 100%, which encode a polypeptide havingcellulolytic enhancing activity.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6comprising a substitution, deletion, and/or insertion of one or more(e.g., several) amino acids. In an embodiment, the number of amino acidsubstitutions, deletions and/or insertions introduced into the maturepolypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 is up to 10,e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be ofa minor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of 1-30 amino acids; smallamino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to about 20-25residues; or a small extension that facilitates purification by changingnet charge or another function, such as a poly-histidine tract, anantigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellulolytic enhancing activity to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The sequenceidentity of essential amino acids can also be inferred from an alignmentwith a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204),and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145;Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellulolytic Enhancing Activity

A polypeptide having cellulolytic enhancing activity of the presentinvention may be obtained from microorganisms of any genus. For purposesof the present invention, the term “obtained from” as used herein inconnection with a given source shall mean that the polypeptide encodedby a nucleotide sequence is produced by the source or by a strain inwhich the nucleotide sequence from the source has been inserted. In apreferred aspect, the polypeptide obtained from a given source issecreted extracellularly.

The polypeptide having cellulolytic enhancing activity of the presentinvention may also be a fungal polypeptide. In one aspect, thepolypeptide is a Thermomyces polypeptide. In another aspect, thepolypeptide is Thermomyces lanuginosus polypeptide having cellulolyticenhancing activity. In a most preferred aspect, the polypeptide is aThermomyces lanuginosus strain NN044973 polypeptide having cellulolyticenhancing activity.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) or DNA samples obtained directly from naturalmaterials (e.g., soil, composts, water, etc.) using the above-mentionedprobes. Techniques for isolating microorganisms and DNA directly fromnatural habitats are well known in the art. A polynucleotide encodingthe polypeptide may then be obtained by similarly screening a genomicDNA or cDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide having cellulolytic enhancing activity of the presentinvention.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pGH61_(—)664which is contained in E. coli CGMCC 4601. In another preferred aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 67 to 869 ofSEQ ID NO: 1. In another more preferred aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding sequencecontained in plasmid pGH61_(—)664 which is contained in E. coli CGMCC4601. The present invention also encompasses nucleotide sequences thatencode polypeptides comprising or consisting of the amino acid sequenceof SEQ ID NO: 2 or the mature polypeptide thereof, which differ from SEQID NO: 1 or the mature polypeptide coding sequence thereof by virtue ofthe degeneracy of the genetic code. The present invention also relatesto subsequences of SEQ ID NO: 1 that encode fragments of SEQ ID NO: 2that have cellulolytic enhancing activity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 3. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pGH61_(—)1590 which is contained in E. coli CGMCC 4602. Inanother preferred aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 3. In anotherpreferred aspect, the nucleotide sequence comprises or consists ofnucleotides 64 to 1036 of SEQ ID NO: 3. In another more preferredaspect, the nucleotide sequence comprises or consists of the maturepolypeptide coding sequence contained in plasmid pGH61_(—)1590 which iscontained in E. coli CGMCC 4602. The present invention also encompassesnucleotide sequences that encode polypeptides comprising or consistingof the amino acid sequence of SEQ ID NO: 4 or the mature polypeptidethereof, which differ from SEQ ID NO: 3 or the mature polypeptide codingsequence thereof by virtue of the degeneracy of the genetic code. Thepresent invention also relates to subsequences of SEQ ID NO: 3 thatencode fragments of SEQ ID NO: 4 that have cellulolytic enhancingactivity.

In another preferred aspect, the nucleotide sequence comprises orconsists of SEQ ID NO: 5. In another more preferred aspect, thenucleotide sequence comprises or consists of the sequence contained inplasmid pGH61_(—)4950 which is contained in E. coli CGMCC 4600. Inanother preferred aspect, the nucleotide sequence comprises or consistsof the mature polypeptide coding sequence of SEQ ID NO: 5. In anotherpreferred aspect, the nucleotide sequence comprises or consists ofnucleotides 67 to 878 of SEQ ID NO: 5. In another more preferred aspect,the nucleotide sequence comprises or consists of the mature polypeptidecoding sequence contained in plasmid pGH61_(—)4950 which is contained inE. coli CGMCC 4600. The present invention also encompasses nucleotidesequences that encode polypeptides comprising or consisting of the aminoacid sequence of SEQ ID NO: 6 or the mature polypeptide thereof, whichdiffer from SEQ ID NO: 5 or the mature polypeptide coding sequencethereof by virtue of the degeneracy of the genetic code. The presentinvention also relates to subsequences of SEQ ID NO: 5 that encodefragments of SEQ ID NO: 6 that have cellulolytic enhancing activity.

The present invention also relates to mutant polynucleotides comprisingor consisting of at least one mutation in the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, in which themutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO:2, SEQ ID NO: 4, or SEQ ID NO: 6, respectively.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Thermomyces, or a related organism andthus, for example, may be an allelic or species variant of thepolypeptide encoding region of the polynucleotide.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofsequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 of at least 70%, at least 75%, at least 80%, at least 81%, atleast 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, more preferably at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, more preferably atleast 95%, and most preferably at least 96%, at least 97%, at least 98%,at least 99%, or 100%, which encode a polypeptide having cellulolyticenhancing activity.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofsequence identity to the mature polypeptide coding sequence of SEQ IDNO: 3 of preferably at least 60%, at least 65%, at least 70%, at least75%, even preferably at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, even more preferably at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, most preferably at least 95%, andeven most preferably at least 96%, at least 97%, at least 98%, at least99%, or 100%, which encode a polypeptide having cellulolytic enhancingactivity.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences that have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 5 of atleast 70%, at least 75%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, more preferably at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, most preferably at least 95%, andeven most preferably at least 96%, at least 97%, at least 98%, at least99%, or 100%, which encode a polypeptide having cellulolytic enhancingactivity.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariant sequence may be constructed on the basis of the polynucleotidepresented as the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 3, or SEQ ID NO: 5, or the cDNA sequences thereof, byintroduction of nucleotide substitutions that do not result in a changein the amino acid sequence of the polypeptide, but which correspond tothe codon usage of the host organism intended for production of theenzyme, or by introduction of nucleotide substitutions that may giverise to a different amino acid sequence. For a general description ofnucleotide substitution, see, e.g., Ford et al., 1991, ProteinExpression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, supra). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for cellulolytic enhancingactivity to identify amino acid residues that are critical to theactivity of the molecule. Sites of substrate-enzyme interaction can alsobe determined by analysis of the three-dimensional structure asdetermined by such techniques as nuclear magnetic resonance analysis,crystallography or photoaffinity labeling (see, e.g., de Vos et al.,1992, supra; Smith et al., 1992, supra; Wlodaver et al., 1992, supra).

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under at leastmedium stringency conditions, or at least medium-high stringencyconditions, preferably high stringency conditions and more preferablyvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) a full-lengthcomplementary strand of (i) or (ii); or allelic variants andsubsequences thereof (Sambrook et al., 1989, supra), as defined herein.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under at least medium stringencyconditions, or at least medium-high stringency conditions, or high orvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (b) isolating the hybridizingpolynucleotide, which encodes a polypeptide having cellulolyticenhancing activity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or more(e.g., several) control sequences that direct the expression of thecoding sequence in a suitable host cell under conditions compatible withthe control sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase Ill,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

In a preferred aspect, the signal peptide comprises or consists of aminoacids 1 to 22 of SEQ ID NO: 2. In another preferred aspect, the signalpeptide coding sequence comprises or consists of nucleotides 1 to 66 ofSEQ ID NO: 1.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 21 of SEQ ID NO: 4. In another preferred aspect, thesignal peptide coding sequence comprises or consists of nucleotides 1 to63 of SEQ ID NO: 3.

In another preferred aspect, the signal peptide comprises or consists ofamino acids 1 to 22 of SEQ ID NO: 6. In another preferred aspect, thesignal peptide coding sequence comprises or consists of nucleotides 1 to66 of SEQ ID NO: 5.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In one aspect, the cell is of the genus Thermomyces. In another aspect,the cell is Thermomyces lanuginosus. In another aspect, the cell isThermomyces lanuginosus NN044973.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention, under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant polynucleotide having at leastone mutation in the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, or SEQ ID NO: 5, wherein the mutant polynucleotide encodesa polypeptide that comprises or consists of the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, respectively; and (b)recovering the polypeptide.

The cells are cultivated in a nutrient medium suitable for production ofthe polypeptide using methods known in the art. For example, the cellsmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentors in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,collection, centrifugation, filtration, extraction, spray-drying,evaporation, or precipitation. In one aspect, the whole fermentationbroth is recovered.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more (e.g., several) expression constructs encodinga polypeptide of the present invention into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the polynucleotide in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying plant cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the polynucleotide encoding a polypeptide ofthe present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide can beintroduced into a particular plant variety by crossing, without the needfor ever directly transforming a plant of that given variety. Therefore,the present invention encompasses not only a plant directly regeneratedfrom cells which have been transformed in accordance with the presentinvention, but also the progeny of such plants. As used herein, progenymay refer to the offspring of any generation of a parent plant preparedin accordance with the present invention. Such progeny may include a DNAconstruct prepared in accordance with the present invention. Crossingresults in the introduction of a transgene into a plant line by crosspollinating a starting line with a donor plant line. Non-limitingexamples of such steps are described in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptidehaving cellulolytic enhancing activity of the present invention underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Cellulolytic Enhancing Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may also beaccomplished by insertion, substitution, or deletion of one or morenucleotides in the gene or a regulatory element required fortranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing thepolynucleotide to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellulolytic enhancing activity in acell, comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation. The present invention also relatesto such double-stranded RNA (dsRNA) molecules, comprising a portion ofthe mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, orSEQ ID NO: 5 for inhibiting expression of a polypeptide in a cell. Whilethe present invention is not limited by any particular mechanism ofaction, the dsRNA can enter a cell and cause the degradation of asingle-stranded RNA (ssRNA) of similar or identical sequences, includingendogenous mRNAs. When a cell is exposed to dsRNA, mRNA from thehomologous gene is selectively degraded by a process called RNAinterference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art, see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The term “heterologous polypeptides” meanspolypeptides that are not native to the host cell, e.g., a variant of anative protein. The host cell may comprise more than one copy of apolynucleotide encoding the native or heterologous polypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellulolytic enhancing activity-free product is of particular interestin the production of eukaryotic polypeptides, in particular fungalproteins such as enzymes. The cellulolytic enhancing activity-deficientcells may also be used to express heterologous proteins ofpharmaceutical interest such as hormones, growth factors, receptors, andthe like. The term “eukaryotic polypeptides” includes not only nativepolypeptides, but also those polypeptides, e.g., enzymes, which havebeen modified by amino acid substitutions, deletions or additions, orother such modifications to enhance activity, thermostability, pHtolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellulolytic enhancing activity that is producedby a method of the present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellulolytic enhancing activity of the composition has been increased,e.g., with an enrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (several) enzymes selected from thegroup consisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

The compositions may be a fermentation broth formulation or a cellcomposition, as described herein. Consequently, the present inventionalso relates to fermentation broth formulations and cell compositionscomprising a polypeptide having cellulolytic enhancing activity of thepresent invention. In some embodiments, the composition is a cell-killedwhole broth containing organic acid(s), killed cells and/or cell debris,and culture medium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compostions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may further comprise one ormore enzyme activities such as acetylxylan esterase,alpha-arabinofuranosidase, alpha-galactosidase, alpha-glucuronidase,amylase, arabinanase, arabinofuranosidase, beta-galactosidase,beta-glucosidase, cellobiohydrolase, endoglucanase,endo-beta-1,3(4)-glucanase, ferrulic acid esterase, galactanase,glucoamylase, glucohydrolase, hybrid peroxidases, with combinedproperties of lignin peroxidases and manganese-dependent peroxidases,laccase, lignin peroxidase, manganese-dependent peroxidases, mannanase,mannan acetyl esterase, mannosidase, pectate lyase, pectin acetylesterase, pectinase lyase, pectin methyl esterase, polygalacturonase,protease, rhamnogalacturonan lyase, rhamnogalacturonan acetyl esterase,rhamnogalacturonase, xylanase, xylogalacturonosidase, xylogalacturonase,xyloglucanase, and xylosidase.

In some embodiments, the cell-killed whole broth or composition includescellulolytic enzymes including, but not limited to, (i) endoglucanases(EG) or 1,4-D-glucan-4-glucanohydrolases (EC 3.2.1.4), (ii)exoglucanases, including 1,4-D-glucan glucanohydrolases (also known ascellodextnnases) (EC 3.2.1.74) and 1,4-D-glucan cellobiohydrolases(exo-cellobiohydrolases, CBH) (EC 3.2.1.91), and (iii) beta-glucosidase(BG) or beta-glucoside glucohydrolases (EC 3.2.1.21).

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following processes forusing the polypeptides having cellulolytic enhancing activity, orcompositions thereof.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention. In oneaspect, the method further comprises recovering the degraded orconverted cellulosic material. Soluble products of degradation orconversion of the cellulosic material can be separated from insolublecellulosic material using a method known in the art such as, forexample, centrifugation, filtration, or gravity settling.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity of the presentinvention. In one aspect, the fermenting of the cellulosic materialproduces a fermentation product. In another aspect, the method furthercomprises recovering the fermentation product from the fermentation.

The methods of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel, potableethanol, and/or platform chemicals (e.g., acids, alcohols, ketones,gases, and the like). The production of a desired fermentation productfrom the cellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using processes conventional in the art.Moreover, the methods of the present invention can be implemented usingany conventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF involves a separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd, L. R.,Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbialcellulose utilization: Fundamentals and biotechnology, Microbiol. Mol.Biol. Reviews 66: 506-577). It is understood herein that any methodknown in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the processes of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics? Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe andZacchi, 2007, Pretreatment of lignocellulosic materials for efficientbioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65;Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility oflignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al.,2005, Features of promising technologies for pretreatment oflignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadehand Karimi, 2008, Pretreatment of lignocellulosic wastes to improveethanol and biogas production: A review, Int. J. of Mol. Sci. 9:1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlockinglow-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment: In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on temperature rangeand addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic material isgenerally only moist during the pretreatment. The steam pretreatment isoften combined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.20020164730). During steam pretreatment, hemicellulose acetyl groups arecleaved and the resulting acid autocatalyzes partial hydrolysis of thehemicellulose to monosaccharides and oligosaccharides. Lignin is removedto only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexplosion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, thecellulosic material is mixed with dilute acid, typically H₂SO₄, andwater to form a slurry, heated by steam to the desired temperature, andafter a residence time flashed to atmospheric pressure. The dilute acidpretreatment can be performed with a number of reactor designs, e.g.,plug-flow reactors, counter-current reactors, or continuouscounter-current shrinking bed reactors (Duff and Murray, 1996, supra;Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999,Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier etal., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or0.1 to 2 wt % acid. The acid is contacted with the cellulosic materialand held at a temperature in the range of preferably 140-200° C., e.g.,165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperatures in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical andbiological treatments for enzymatic/microbial conversion of cellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose to fermentable sugars, such as glucose, cellobiose,xylose, xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides. The hydrolysis is performed enzymatically by an enzymecomposition as described herein in the presence of a polypeptide havingcellulolytic enhancing activity of the present invention. The enzymecomponents of the compositions can be added simultaneously orsequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme components, i.e.,optimal for the enzyme components. The hydrolysis can be carried out asa fed batch or continuous process where the cellulosic material is fedgradually to, for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 5.0 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt %, e.g., about 10 toabout 40 wt % or about 20 to about 30 wt %.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (e.g., several) enzymes selectedfrom the group consisting of an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase and a polypeptidehaving cellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase,and a polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises a cellobiohydrolase, abeta-glucosidase, and a polypeptide having cellulolytic enhancingactivity. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the enzyme composition comprises an endoglucanase, acellobiohydrolase, a beta-glucosidase, and a polypeptide havingcellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase).

In another aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the processes of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and a polypeptide having cellulolyticenhancing activity depend on several factors including, but not limitedto, the mixture of cellulolytic and/or hemicellulolytic enzymecomponents, the cellulosic material, the concentration of cellulosicmaterial, the pretreatment(s) of the cellulosic material, temperature,time, pH, and inclusion of fermenting organism (e.g., yeast forSimultaneous Saccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to the cellulosic material is about 0.01to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 toabout 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosicmaterial.

In another aspect, an effective amount of a polypeptide havingcellulolytic enhancing activity to cellulolytic or hemicellulolyticenzyme is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g,about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 toabout 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g perg of cellulolytic or hemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material (collectively hereinafter“polypeptides having enzyme activity”) can be derived or obtained fromany suitable origin, including, bacterial, fungal, yeast, plant, ormammalian origin. The term “obtained” also means herein that the enzymemay have been produced recombinantly in a host organism employingmethods described herein, wherein the recombinantly produced enzyme iseither native or foreign to the host organism or has a modified aminoacid sequence, e.g., having one or more (e.g., several) amino acids thatare deleted, inserted and/or substituted, i.e., a recombinantly producedenzyme that is a mutant and/or a fragment of a native amino acidsequence or an enzyme produced by nucleic acid shuffling processes knownin the art. Encompassed within the meaning of a native enzyme arenatural variants and within the meaning of a foreign enzyme are variantsobtained recombinantly, such as by site-directed mutagenesis orshuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes NS),CELLIC® CTec2 (Novozymes NS), CELLIC® Ctec3 (Novozymes NS), CELLUCLAST™(Novozymes NS), NOVOZYM™ 188 (Novozymes NS), CELLUZYME™ (Novozymes NS),CEREFLO™ (Novozymes NS), and ULTRAFLO™ (Novozymes NS), ACCELERASE™(Genencor Int.), LAMINEX™ (Genencor Int.), SPEZYME™ CP (Genencor Int.),FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (RohmGmbH), FIBREZYME® LDI (Dyadic International, Inc.), FIBREZYME® LBR(Dyadic International, Inc.), or VISCOSTAR® 150L (Dyadic International,Inc.). The cellulase enzymes are added in amounts effective from about0.001 to about 5.0 wt % of solids, e.g., about 0.025 to about 4.0 wt %of solids or about 0.005 to about 2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the processesof the present invention, include, but are not limited to, anAcidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186;U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (WO05/093050); and Thermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GENBANK™ accession no. M15665),Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22), Trichoderma reesei Cel5A endoglucanase II (GENBANK™ accessionno. M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563, GENBANK™ accession no. AB003694),Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228, GENBANK™ accession no. Z33381), Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439), Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14), Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381), Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107), Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703), Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_(—)324477), Humicolainsolens endoglucanase V, Myceliophthora thermophila CBS 117.65endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6Bendoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase,Thielavia terrestris NRRL 8126 CEL7C endoglucanase, Thielavia terrestrisNRRL 8126 CEL7E endoglucanase, Thielavia terrestris NRRL 8126 CEL7Fendoglucanase, Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase, and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielaviaterrestris cellobiohydrolase II (CEL6A, WO 2006/074435), Trichodermareesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637.

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,648,263, and U.S. Pat. No. 5,686,593.

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a soluble activating divalent metalcation according to WO 2008/151043, e.g., manganese sulfate.

In another aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover(PCS).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicycliccompounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of the nitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ toabout 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁶ toabout 1, about 10⁻⁶ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, aneffective amount of such a compound described above is about 0.1 μM toabout 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide can be produced by treating a lignocellulose orhemicellulose material (or feedstock) by applying heat and/or pressure,optionally in the presence of a catalyst, e.g., acid, optionally in thepresence of an organic solvent, and optionally in combination withphysical disruption of the material, and then separating the solutionfrom the residual solids. Such conditions determine the degree ofcellulolytic enhancement obtainable through the combination of liquorand a GH61 polypeptide during hydrolysis of a cellulosic substrate by acellulase preparation. The liquor can be separated from the treatedmaterial using a method standard in the art, such as filtration,sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about 1g, about 10⁻⁶ to about 1 g, about 10⁻⁶ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes NS),CELLIC® HTec (Novozymes NS), CELLIC® HTec2 (Novozymes NS), VISCOZYME®(Novozymes NS), ULTRAFLO® (Novozymes NS), PULPZYME® HC (Novozymes NS),MULTIFECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor), ACCELLERASE®XC (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM),DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L. (BiocatalystsLimit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit, Wales, UK).

Examples of xylanases useful in the processes of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), andTrichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the processes of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt accession number Q7SOW4), Trichodermareesei (UniProtKB/TrEMBL accession number Q92458), and Talaromycesemersonii (SwissProt accession number Q8X212).

Examples of acetylxylan esterases useful in the processes of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprotaccession number Q2GWX4), Chaetomium gracile (GeneSeqP accession numberAAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocreajecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880),Neurospora crassa (UniProt accession number q7s259), Phaeosphaerianodorum (Uniprot accession number QOUHJ1), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in theprocesses of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt Accession number A1D9T4), Neurosporacrassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum(WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO2010/065448).

Examples of arabinofuranosidases useful in the processes of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP accession number AAR94170), Humicolainsolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus(WO 2006/114094).

Examples of alpha-glucuronidases useful in the processes of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt accession number alcc12), Aspergillusfumigatus (SwissProt accession number Q4WW45), Aspergillus niger(Uniprot accession number Q96WX9), Aspergillus terreus (SwissProtaccession number QOCJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt accession number Q8X211), and Trichoderma reesei (Uniprotaccession number Q99024).

The polypeptides having enzyme activity used in the processes of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, CA, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol, 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Preferred yeastincludes strains of Candida, Kluyveromyces, and Saccharomyces, e.g.,Candida sonorensis, Kluyveromyces marxianus, and Saccharomycescerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces marxianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia. In another more preferred aspect, the yeast is a Pichiastipitis. In another preferred aspect, the yeast is a Saccharomyces spp.In a more preferred aspect, the yeast is Saccharomyces cerevisiae. Inanother more preferred aspect, the yeast is Saccharomyces distaticus. Inanother more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacillus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia coli. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces marxianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximate 10⁵ to 10¹², preferably from approximate 10⁷ to 10¹⁰,especially approximate 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe processes of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is n-butanol. In another more preferred aspect, the alcohol isisobutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is methanol. In another morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanediol. In another more preferred aspect, thealcohol is ethylene glycol. In another more preferred aspect, thealcohol is glycerin. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is1,3-propanediol. In another more preferred aspect, the alcohol issorbitol. In another more preferred aspect, the alcohol is xylitol. See,for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Detergent Compositions

The polypeptides having cellulolytic enhancing activity of the presentinvention may be added to and thus become a component of a detergentcomposition.

The detergent composition of the present invention may be formulated,for example, as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide of the invention. The detergentadditive as well as the detergent composition may comprise one or more(e.g., several) enzymes such as a protease, lipase, cutinase, anamylase, carbohydrase, cellulase, pectinase, mannanase, arabinase,galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

In general the properties of the selected enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263,U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include CELLUZYME™, and CAREZYME™(Novozymes NS), CLAZINASE™, and PURADAX HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Proteases:

Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically modified or proteinengineered mutants are included. The protease may be a serine proteaseor a metalloprotease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases are subtilisins,especially those derived from Bacillus, e.g., subtilisin Novo,subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168(described in WO 89/06279). Examples of trypsin-like proteases aretrypsin (e.g., of porcine or bovine origin) and the Fusarium proteasedescribed in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and274.

Preferred commercially available protease enzymes include ALCALASE™SAVINASE™, PRIMASE™, DURALASE™, ESPERASE™, and KANNASE™ (Novozymes NS),MAXATASE™, MAXACAL™, MAXAPEM™, PROPERASE™, PURAFECT™, PURAFECT OXP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases:

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g., fromH. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis(Dartois et al., 1993, Biochemica et Biophysica Acta, 1131: 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipase enzymes include LIPOLASE™ andLIPOLASE ULTRA™ (Novozymes NS).

Amylases:

Suitable amylases (α and/or β) include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Amylases include, for example, α-amylases obtained from Bacillus, e.g.,a special strain of Bacillus licheniformis, described in more detail inGB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are DURAMYL™, TERMAMYL™, FUNGAMYL™ andBAN™ (Novozymes NS), RAPIDASE™ and PURASTAR™ (from GenencorInternational Inc.).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include GUARDZYME™ (Novozymes NS).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more (e.g., several)enzymes, or by adding a combined additive comprising all of theseenzymes. A detergent additive of the invention, i.e., a separateadditive or a combined additive, can be formulated, for example, as agranulate, liquid, slurry, etc. Preferred detergent additiveformulations are granulates, in particular non-dusting granulates,liquids, in particular stabilized liquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more (e.g., several)surfactants, which may be non-ionic including semi-polar and/or anionicand/or cationic and/or zwitterionic. The surfactants are typicallypresent at a level of from 0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates, or layered silicates (e.g., SKS-6 fromHoechst).

The detergent may comprise one or more (e.g., several) polymers.Examples are carboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol), poly(vinyl alcohol), poly(vinylpyridine-N-oxide),poly(vinylimidazole), polycarboxylates such as polyacrylates,maleic/acrylic acid copolymers, and lauryl methacrylate/acrylic acidcopolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions, any enzyme may be added in an amountcorresponding to 0.01-100 mg of enzyme protein per liter of wash liquor,preferably 0.05-5 mg of enzyme protein per liter of wash liquor, inparticular 0.1-1 mg of enzyme protein per liter of wash liquor.

In the detergent compositions, a polypeptide of the present inventionhaving cellulolytic enhancing activity may be added in an amountcorresponding to 0.001-100 mg of protein, preferably 0.005-50 mg ofprotein, more preferably 0.01-25 mg of protein, even more preferably0.05-10 mg of protein, most preferably 0.05-5 mg of protein, and evenmost preferably 0.01-1 mg of protein per liter of wash liquor.

A polypeptide of the invention having cellulolytic enhancing activitymay also be incorporated in the detergent formulations disclosed in WO97/07202, which is hereby incorporated by reference.

Signal Peptides

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to22 of SEQ ID NO: 2, amino acids 1 to 21 of SEQ ID NO: 4, or amino acids1 to 22 of SEQ ID NO: 6. The present invention also relates to nucleicacid constructs comprising a gene encoding a protein, which is operablylinked to the signal peptide. The protein is preferably foreign to thesignal peptide.

In a preferred aspect, the polynucleotide encoding the signal peptidecomprises or consists of nucleotides 1 to 66 of SEQ ID NO: 1,nucleotides 1 to 63 of SEQ ID NO: 3, or nucleotides 1 to 66 of SEQ IDNO: 5.

The present invention also relates to recombinant expression vectorscomprising such polynucleotides and recombinant host cells comprisingsuch nucleic acid constructs.

The present invention also relates to methods of producing a proteincomprising (a) cultivating a recombinant host cell comprising suchpolynucleotide; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Strains

Thermomyces lanuginosus strain NN044973 was isolated in 1998 from Yunnanprovince, China.

Media

PDA medium was composed of 39 grams of potato dextrose agar anddeionized water to 1 liter.

YPG medium was composed of 0.4% of yeast extract, 0.1% of KH₂PO₄, 0.05%of MgSO₄.7H₂O, and 1.5% glucose in deionized water.

Minimal medium plates were composed of 6 g of NaNO₃, 0.52 g of KCl, 1.52g of KH₂PO₄, 1 ml of COVE trace elements solution, 20 g of Noble agar,20 ml of 50% glucose, 2.5 ml of MgSO₄.7H₂O, 20 ml of a 0.02% biotinsolution, and deionized water to 1 liter.

YPM medium was composed of 1% of yeast extract, 2% of peptone and 2% ofmaltose in deionized water.

Example 1 Thermomyces lagnunosis Genomic DNA Extraction

Thermomyces lagnunosis strain NN044973 was inoculated onto a PDA plateand incubated for 3 days at 45° C. in the darkness. Several mycelia-PDAplugs were inoculated into 500 ml shake flasks containing 100 ml of YPGmedium. The flasks were incubated for 3 days at 45° C. with shaking at160 rpm. The mycelia were collected by filtration through MIRACLOTH®(Calbiochem, La Jolla, Calif., USA) and frozen in liquid nitrogen.Frozen mycelia were ground, by mortar and pestle, to a fine powder, andgenomic DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Inc.,Valencia, Calif., USA).

Example 2 Genome Sequencing and Assembly

The extracted genomic DNA samples were delivered to Beijing GenomeInstitute (BGI, Shenzhen, China) for genome sequencing using ILLUMINA®GA2 System (Illumina, Inc., San Diego, Calif., USA). The raw reads wereassembled at BGI using SOAPdenovo (Li et al., 2010, Genome Research20(2): 265-72). The assembled sequences were analyzed using standardbioinformatics methods for gene finding and functional prediction.Briefly, geneID (Parra et al., 2000, Genome Research 10(4): 511-515) wasused for gene prediction. Blastall version 2.2.10 (Altschul et al.,1990, J. Mol. Biol. 215 (3): 403-410; National Center for BiotechnologyInformation (NCBI), Bethesda, Md., USA) and HMMER version 2.1.1(National Center for Biotechnology Information (NCBI), Bethesda, Md.,USA) were used to predict function based on structural homology. Thefamily GH61 polypeptides were identified directly by analysis of theBlast results. The Agene program (Munch and Krogh, 2006, BMCBioinformatics 7: 263) and SignalP program (Nielsen et al., 1997,Protein Engineering 10: 1-6) were used to identify starting codons.SignalP was further used to predict the signal peptides.

Example 3 Cloning of the Thermomyces lanuginosus GH61(1) Gene fromGenomic DNA

Based on a Thermomyces lanuginosus GH61 gene sequence as identified inExample 2, oligonucleotide primers, shown below, were designed toamplify the gene from genomic DNA of Thermomyces lanuginosus. AnIN-FUSION™ CF Dry-down PCR Cloning Kit (Clontech Laboratories, Inc.,Mountain View, Calif., USA) was used to clone the fragment directly intothe expression vector pPFJO355, without the need for restrictiondigestion and ligation.

Sense primer: (SEQ ID NO: 7) 5′-ACACAACTGGGGATCC ACCATGAAGGGCTCCAGCGCTG-3′ Antisense primer: (SEQ ID NO: 8)5′-GTCACCCTCTAGATCT CTCAACGCACCATGTACTCGTCTC-3′

Lowercase characters of the forward primer represent the coding regionsof the gene and lowercase characters of the reverse primer represent thecoding region or the flanking region of the gene, while capitalizedparts were homologous to the insertion sites of pPFJO355 vector whichhas been described in US2010306879.

The expression vector pPFJO355 contains the TAKA-amylase promoterderived from Aspergillus oryzae and the Aspergillus niger glucoamylaseterminator elements. Furthermore pPFJO355 has pUC18 derived sequencesfor selection and propagation in E. coli, and a pyrG gene, which encodesan orotidine decarboxylase derived from Aspergillus nidulans forselection of a transformant of a pyrG mutant Aspergillus strain.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Thermomyces lanuginosus genomic DNA, 10 μl of 5× HFBuffer, 1.5 μl of DMSO, 2 μl of 2.5 mM each of dATP, dTTP, dGTP, anddCTP, and 1 unit of PHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy,Espoo, Finland) in a final volume of 50 μl. The amplification wasperformed using a Peltier Thermal Cycler (MJ Research, Inc., Waltham,Mass., USA) programmed for denaturing at 98° C. for 1 minutes; 5 cyclesof denaturing at 98° C. for 15 seconds, annealing at 65° C. for 30seconds, with 1° C. decreasing per cycle and elongation at 72° C. for 60seconds; and another 25 cycles each at 98° C. for 15 seconds andannealing at 60° C. for 30 seconds; elongation at 72° C. for 60 seconds;final extension at 72° C. for 10 minutes. The heat block then went to a4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 90 mM Tris-borate and 1 mM EDTA (TBE) buffer where an approximate900 bp product band was excised from the gel, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare,Buckinghamshire, UK) according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ Dry-down PCR Cloning kit resulting in pGH61_(—)664 in whichtranscription of the Thermomyces lanuginosus GH61 gene was under thecontrol of a promoter from the gene for Aspergillus oryzaealpha-amylase. The cloning operation was according to the manufacturer'sinstruction. In brief, 30 ng of pPFJO355 digested with Barn I and BglII, and 50 ng of the Thermomyces lanuginosus GH61 gene purified PCRproduct were added to a reaction vial and resuspended in a final volumeof 5 μl with addition of deionized water. The reaction was incubated at37° C. for 15 minutes and then 50° C. for 15 minutes. Five μl of thereaction were used to transform E. coli TOP10 competent cells (TIANGENBiotech (Beijing) Co. Ltd., Beijing, China). An E. coli transformantcontaining pGH61_(—)664 was detected by colony PCR and plasmid DNA wasprepared using a QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia,Calif., USA). The inserted GH61 gene was confirmed by a DNA sequencingcompany (SinoGenoMax, Beijing). E. coli

TOP10 strain, containing pGH61_(—)664, was deposited with China GeneralMicrobiological Culture Collection Center (CGMCC) in Beijing on Feb. 23,2011, and assigned accession number as CGMCC 4601.

Example 4 Characterization of the Genomic DNA Encoding GH61(1)Polypeptide

The genomic DNA sequence and deduced amino acid sequence of aThermomyces lanuginosus GH61 polypeptide coding sequence are shown inSEQ ID NO: 1 and SEQ ID NO: 2, respectively. The coding sequence is 872bp including the stop codon which is interrupted by 1 intron of 53 bp(nucleotides 335 to 387). The encoded predicted protein is 272 aminoacids. Using the SignalP program (Nielsen et al., 1997, supra), a signalpeptide of 22 residues was predicted. The predicted mature proteincontains 250 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with gap open penalty of 10, gap extension penaltyof 0.5, and the EBLOSUM62 matrix. The alignment showed that the deducedamino acid sequence of the Thermomyces lanuginosus genomic DNA encodinga GH61 polypeptide shares 68.0% identity (excluding gaps) to a putativeendo-1,4-beta-glucanase from Aspergillus fumigatus (UNIPROT:B0XZE1_ASPFC).

Example 5 Expression of Thermomyces lanuginosus GH61(1) Gene inAspergillus oryzae

Aspergillus oryzae HowB101 (described in WO95/35385 example 1)protoplasts were prepared according to the method of Christensen et al.,1988, Bio/Technology 6: 1419-1422. Three μg of pGH61_(—)644 were used totransform Aspergillus oryzae HowB101.

The transformation of Aspergillus oryzae HowB101 with pGH61_(—)664yielded about 20 transformants. Four transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C., 150 rpm. After 3 daysincubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NuPAGE® Novex 4-12% Bis-Tris Gel with2-(N-morpholino)ethanesulfonic acid (MES) (Invitrogen Corporation,Carlsbad, Calif., USA) according to the manufacturer's instructions. Theresulting gel was stained with INSTANT BLUE™ (Expedeon Ltd., BabrahamCambridge, UK). SDS-PAGE profiles of the cultures showed that themajority of the transformants had a band of approximate 40 kDa. Theexpression strain was designated as O5MMD.

A slant of one transformant was washed with 10 ml of YPM medium andinoculated into a 2 liter flask containing 400 ml of YPM medium togenerate broth for characterization of the enzyme. The culture washarvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane(Millipore, Bedford, Mass., USA).

Example 6 Hydrolysis of Pretreated Corn Stover is Enhanced byThermomyces Lanuginosus GH61(1) Polypeptide Having CellulolyticEnhancing Activity

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 0.048 g sulfuric acid/g drybiomass at 190° C. and 25% w/w dry solids for around 1 minute. Thewater-insoluble solids in the pretreated corn stover (PCS) contained 54%cellulose. Cellulose was determined by a two-stage sulfuric acidhydrolysis with subsequent analysis of sugars by high performance liquidchromatography (HPLC) using NREL Standard Analytical Procedure #002.Prior to enzymatic hydrolysis, the PCS was washed by water and groundusing a Multi Utility Grinder (Inno Concepts Inc., Roswell, Ga., USA)and sieved through a 450 μm screen by AS200 (Retsch, Haman, Germany).

The hydrolysis of pretreated corn stover was conducted using 1.8 ml,96-deep well plates (Beckman Instruments INC. Fullerton, USA) containinga total reaction mass of 1 g. The hydrolysis was performed with 7.8%total solids of washed pretreated corn stover, equivalent to 54 mg ofcellulose per ml, in 5 mM manganese sulfate—20 mM sodium acetate pH 5.0buffer containing a Trichoderma reesei base cellulase mixture(CELLUCLAST® supplemented with Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)available from Novozymes NS, Bagsvaerd, Denmark; the cellulasecomposition is designated herein in the Examples as “Trichoderma reeseicellulase composition”) at 2 mg per g of cellulose. Thermomyceslanuginosus GH61(1) polypeptide having cellulolytic enhancing activitywas separately added to the base cellulase mixture at ranging from 0 to90% of the concentration of base cellulase mixture. Plates were cappedusing a Capmat (Beckman Coulter, USA) and incubated at 50° C. and 60° C.for 118 hours with shaking at 150 rpm.

After 118 hours of incubation, 100 μl aliquots were removed and theextent of hydrolysis was assayed by high-performance liquidchromatography (HPLC) using the protocol described below.

For HPLC analysis, samples were filtered with a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. The sugar concentrationsof samples diluted in 5 mM H₂SO₄ were measured using a 4.6×250 mmAMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) by elution with 5 mM H₂SO₄ at a flow rate of 0.6 ml per minute at60° C. for 11 minutes, and quantification by integration of the glucoseand cellobiose signal from refractive index detection (CHEMSTATION®,AGILENT® 1200 HPLC, Agilent Technologies, Santa Clara, Calif., USA)calibrated by pure sugar samples. The resultant equivalents were used tocalculate the percentage of cellulose conversion for each reaction. Theextent of each hydrolysis was determined as the fraction of totalcellulose converted to cellobiose+glucose, and was not corrected forsoluble sugars present in pretreated corn stover liquor.

Measured sugar concentrations were adjusted for the appropriate dilutionfactor. Glucose and cellobiose were chromatographically separated andintegrated and their respective concentrations determined independently.However, to calculate total conversion the glucose and cellobiose valueswere combined. Fractional hydrolysis is reported as the overall massconversion to [glucose+cellobiose]/[total cellulose].

The data demonstrated enhancement of hydrolysis by addition of theThermomyces lanuginosus GH61(1) polypeptide having cellulolyticenhancing activity. The addition of the GH61(1) polypeptide at 23%, 37%and 47% (w/w) enhanced hydrolysis by 18.4%, 21.0% and 16.3% glucanconversion in 118 hours of hydrolysis at 50° C., and enhanced hydrolysisby 51.4%, 27.4% and 40.1% glucan conversion in 118 hours of hydrolysisat 60° C.

Example 7 Cloning of the Thermomyces lanuginosus GH61(2) Gene fromGenomic DNA

Based on a Thermomyces lanuginosus GH61 gene sequence as identified inExample 2, oligonucleotide primers, shown below, were designed toamplify the gene from genomic DNA of Thermomyces lanuginosus prepared inExample 1. An IN-FUSION™ CF Dry-down PCR Cloning Kit (ClontechLaboratories, Inc., Mountain View, Calif., USA) was used to clone thefragment directly into the expression vector pPFJO355, without the needfor restriction digestion and ligation.

Sense primer: (SEQ ID NO: 9) 5′-ACACAACTGGGGATCC ACCATGGCATTCTCTACGGTTACAGTTTT TGTTAC-3′ Antisense primer: (SEQ ID NO: 10)5′-GTCACCCTCTAGATCT AATGAGAGAGCATATCCATAACCGCAT-3′

Lowercase characters of the forward primer represent the coding regionsof the gene and lowercase characters of the reverse primer represent thecoding region or the flanking region of the gene, while capitalizedparts were homologous to the insertion sites of pPFJO355 vector whichhas been described in US2010306879.

The expression vector pPFJO355 contains the TAKA-amylase promoterderived from Aspergillus oryzae and the Aspergillus niger glucoamylaseterminator elements. Furthermore pPFJO355 has pUC18 derived sequencesfor selection and propagation in E. coli, and a pyrG gene, which encodesan orotidine decarboxylase derived from Aspergillus nidulans forselection of a transformant of a pyrG mutant Aspergillus strain.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Thermomyces lanuginosus genomic DNA, 10 μl of 5× HFBuffer, 1.5 μl of DMSO, 2 μl of 2.5 mM each of dATP, dTTP, dGTP, anddCTP, and 1 unit of PHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy,Espoo, Finland) in a final volume of 50 μl. The amplification wasperformed using a Peltier Thermal Cycler (MJ Research, Inc., Waltham,Mass., USA) programmed for denaturing at 98° C. for 1 minute; 5 cyclesof denaturing at 98° C. for 15 seconds, annealing at 65° C. for 30seconds, with 1° C. decreasing per cycle and elongation at 72° C. for 60seconds; and another 25 cycles each at 98° C. for 15 seconds andannealing at 60° C. for 30 seconds; elongation at 72° C. for 60 seconds;and a final extension at 72° C. for 10 minutes. The heat block then wentto a 4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 90 mM Tris-borate and 1 mM EDTA (TBE) buffer where an approximate1.0 kb product band was excised from the gel, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare,Buckinghamshire, UK) according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ Dry-down PCR Cloning kit resulting in pGH61_(—)1590 in whichtranscription of the Thermomyces lanuginosus GH61 gene was under thecontrol of a promoter from the gene for Aspergillus oryzaealpha-amylase. The cloning operation was according to the manufacturer'sinstruction. In brief, 30 ng of pPFJO355 digested with Barn I and BglII, and 50 ng of the Thermomyces lanuginosus GH61 gene purified PCRproduct were added to a reaction vial and the reaction powder wasresuspended in a final volume of 5 μl with addition of deionized water.The reaction was incubated at 37° C. for 15 minutes and then 50° C. for15 minutes. Five μl of the reaction were used to transform E. coli TOP10competent cells (TIANGEN Biotech (Beijing) Co. Ltd., Beijing, China). AnE. coli transformant containing pGH61_(—)1590 was detected by colony PCRand plasmid DNA was prepared using a QIAprep Spin Miniprep Kit (QIAGENInc., Valencia, Calif., USA). The inserted GH61 gene was confirmed by aDNA sequencing company (SinoGenoMax, Beijing). E. coli TOP10 strain,containing pGH61_(—)1590, was deposited with China GeneralMicrobiological Culture Collection Center (CGMCC) in Beijing on Feb. 23,2011, and assigned accession number as CGMCC 4602.

Example 8 Characterization of the Genomic DNA Encoding GH61(2)Polypeptide

The genomic DNA sequence and deduced amino acid sequence of aThermomyces lanuginosus GH61 polypeptide coding sequence are shown inSEQ ID NO: 3 and SEQ ID NO: 4, respectively. The coding sequence is 1039bp including the stop codon which is interrupted by 1 intron of 55 bp(nucleotides 111 to 165). The encoded predicted protein is 327 aminoacids. Using the SignalP program (Nielsen et al., 1997, supra), a signalpeptide of 21 residues was predicted. The predicted mature proteincontains 306 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with gap open penalty of 10, gap extension penaltyof 0.5, and the EBLOSUM62 matrix. The alignment showed that the deducedamino acid sequence of the Thermomyces lanuginosus genomic DNA encodinga GH61 polypeptide shares 49.2% identity (excluding gaps) to aThermoascus aurantiacus GH61 B protein (WO 2010065830).

Example 9 Expression of Thermomyces lanuginosus GH61(2) Gene inAspergillus oryzae

Aspergillus oryzae HowB101 (described in WO95/35385 example 1)protoplasts were prepared according to the method of Christensen et al.,1988, Bio/Technology 6: 1419-1422. Three μg of pGH61_(—)1590 were usedto transform Aspergillus oryzae HowB101.

The transformation of Aspergillus oryzae HowB101 with pGH61_(—)1590yielded about 20 transformants. Four transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C., 150 rpm. After 3 daysincubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NuPAGE® Novex 4-12% Bis-Tris Gel with2-(N-morpholino)ethanesulfonic acid (MES) (Invitrogen Corporation,Carlsbad, Calif., USA) according to the manufacturer's instructions. Theresulting gel was stained with INSTANT BLUE™ (Expedeon Ltd., BabrahamCambridge, UK). SDS-PAGE profiles of the cultures showed that themajority of the transformants had a smear of approximate 60 kDa. Theexpression strain was designated as O5MME.

A slant of one transformant was washed with 10 ml of YPM medium andinoculated into a 2 liter flask containing 400 ml of YPM medium togenerate broth for characterization of the enzyme. The culture washarvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane(Millipore, Bedford, Mass., USA).

Example 10 Purification of Recombinant Thermomyces lanuginosus GH61(2)from Aspergillus oryzae

3200 ml supernatant of the recombinant transformant prepared asdescribed in Example 9 was precipitated with ammonium sulfate (80%saturation) and re-dissolved in 50 ml of 20 mM

Tris-HCl buffer, pH6.5, then dialyzed against the same buffer andfiltered through a 0.45 mm filter, the final volume was 110 ml. Thesolution was applied to a 40 ml Q SEPHAROSE® Fast Flow column (GEHealthcare, Buckinghamshire, UK) equilibrated in 20 mM Tris-HCl buffer,pH6.5, and the proteins was eluted with a linear NaCl gradient(0-0.25M). Fractions were evaluated by SDS-PAGE (NP0336BOX, NuPAGE®Novex 4-12% Bis-Tris GEL 1.5 mM15 W). Fractions containing a band ofapproximate 35 kDa were pooled. Then the pooled solution wasconcentrated by ultrafiltration.

Example 11 Hydrolysis of Pretreated Corn Stover is Enhanced byThermomyces lanuginosus GH61(2) Polypeptide Having CellulolyticEnhancing Activity

The hydrolysis of dilute acid pretreated corn stover (PCS) was conductedusing 1.8 mL, 96-deep well plates (Beckman Instruments INC. Fullerton,USA) containing a total reaction mass of 1 g. The hydrolysis wasperformed with 7.2% total solids of washed, pretreated corn stover,equivalent to 50 mg of cellulose per ml, in 5 mM manganese sulfate—20 mMsodium acetate pH 5.0 buffer containing a Trichoderma reesei cellulasecomposition (CELLUCLAST® supplemented with Aspergillus oryzaebeta-glucosidase (recombinantly produced in Aspergillus oryzae accordingto WO 02/095014) available from Novozymes NS, Bagsvaerd, Denmark; thecellulase composition is designated herein in the Examples as“Trichoderma reesei cellulase composition”) at 2 mg per g of cellulose.Thermomyces lanuginosus GH61(2) polypeptide having cellulolyticenhancing activity was added at concentrations of 23% (w/w) of totalprotein. Plates were capped using a Capmat (Beckman Coulter, USA) andincubated at 60° C. for 44 hours with shaking at 150 rpm. Allexperiments were performed in triplicate.

After 44 hours of incubation, 100 μl aliquots were removed and theextent of hydrolysis was assayed by high-performance liquidchromatography (HPLC) using the protocol described below.

For HPLC analysis, samples were filtered with a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. The sugar concentrationsof samples diluted in 5 mM H₂SO₄ were measured using a 4.6×250 mmAMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) by elution with 5 mM H₂SO₄ at a flow rate of 0.6 ml per minute at60° C. for 11 minutes, and quantification by integration of the glucoseand cellobiose signal from refractive index detection (CHEMSTATION®,AGILENT® 1200 HPLC, Agilent Technologies, Santa Clara, Calif., USA)calibrated by pure sugar samples. The resultant equivalents were used tocalculate the percentage of cellulose conversion for each reaction. Theextent of each hydrolysis was determined as the fraction of totalcellulose converted to cellobiose+glucose, and was not corrected forsoluble sugars present in pretreated corn stover liquor.

Measured sugar concentrations were adjusted for the appropriate dilutionfactor. Glucose and cellobiose were chromatographically separated andintegrated and their respective concentrations determined independently.However, to calculate total conversion the glucose and cellobiose valueswere combined. Fractional hydrolysis is reported as the overall massconversion to [glucose+cellobiose]/[total cellulose]. Triplicate datapoints were averaged.

The data demonstrated enhancement of hydrolysis by addition of theThermomyces lanuginosus GH61(2) polypeptide having cellulolyticenhancing activity. Addition of the GH61 polypeptide at 23% (w/w)enhanced hydrolysis by 3.99%, from 6.51% to 6.77% glucan conversion in44 hours of hydrolysis.

Example 12 Cloning of the Thermomyces lanuginosus GH61(3) Gene fromGenomic DNA

Based on a Thermomyces lanuginosus GH61 gene sequence as identified inExample 2, oligonucleotide primers, shown below, were designed toamplify the gene from genomic DNA of Thermomyces lanuginosus prepared inExample 1. An IN-FUSION™ CF Dry-down PCR Cloning Kit (ClontechLaboratories, Inc., Mountain View, Calif., USA) was used to clone thefragment directly into the expression vector pPFJO355, without the needfor restriction digestion and ligation.

Sense primer: (SEQ ID NO: 11) 5′-ACACAACTGGGGATCC ACCATGAAAGGCTCCACCACTGCG-3′ Antisense primer: (SEQ ID NO: 12)5′-GTCACCCTCTAGATCT CAGCGGTAGCAAGCATTCGACT-3′

Lowercase characters of the forward primer represent the coding regionsof the gene and lowercase characters of the reverse primer represent thecoding region or the flanking region of the gene, while capitalizedparts were homologous to the insertion sites of pPFJO355 vector whichhas been described in US2010306879.

The expression vector pPFJO355 contains the TAKA-amylase promoterderived from Aspergillus oryzae and the Aspergillus niger glucoamylaseterminator elements. Furthermore pPFJO355 has pUC18 derived sequencesfor selection and propagation in E. coli, and a pyrG gene, which encodesan orotidine decarboxylase derived from Aspergillus nidulans forselection of a transformant of a pyrG mutant Aspergillus strain.

Twenty picomoles of each of the primers above were used in a PCRreaction composed of Thermomyces lanuginosus genomic DNA, 10 μl of 5× HFBuffer, 1.5 μl of DMSO, 2 μl of 2.5 mM each of dATP, dTTP, dGTP, anddCTP, and 1 unit of PHUSION™ High-Fidelity DNA Polymerase (Finnzymes Oy,Espoo, Finland) in a final volume of 50 μl. The amplification wasperformed using a Peltier Thermal Cycler (MJ Research, Inc., Waltham,Mass., USA) programmed for denaturing at 98° C. for 1 minutes; 5 cyclesof denaturing at 98° C. for 15 seconds, annealing at 65° C. for 30seconds, with 1° C. decreasing per cycle and elongation at 72° C. for 60seconds; and another 25 cycles each at 98° C. for 15 seconds andannealing at 60° C. for 30 seconds; elongation at 72° C. for 60 seconds;final extension at 72° C. for 10 minutes. The heat block then went to a4° C. soak cycle.

The reaction products were isolated by 1.0% agarose gel electrophoresisusing 90 mM Tris-borate and 1 mM EDTA (TBE) buffer where an approximate900 bp product band was excised from the gel, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare,Buckinghamshire, UK) according to the manufacturer's instructions.

Plasmid pPFJO355 was digested with Barn I and Bgl II, isolated by 1.0%agarose gel electrophoresis using TBE buffer, and purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit according to themanufacturer's instructions.

The gene fragment and the digested vector were ligated together using anIN-FUSION™ Dry-down PCR Cloning kit resulting in pGH61_(—)4950 in whichtranscription of the Thermomyces lanuginosus GH61 gene was under thecontrol of a promoter from the gene for Aspergillus oryzaealpha-amylase. The cloning operation was according to the manufacturer'sinstruction. In brief, 30 ng of pPFJO355 digested with Barn I and BglII, and 50 ng of the Thermomyces lanuginosus GH61 gene purified PCRproduct were added to a reaction vial and resuspended in a final volumeof 5 μl with addition of deionized water. The reaction was incubated at37° C. for 15 minutes and then 50° C. for 15 minutes. Five μl of thereaction were used to transform E. coli TOP10 competent cells (TIANGENBiotech (Beijing) Co. Ltd., Beijing, China). An E. coli transformantcontaining pGH61_(—)4950 was detected by colony PCR and plasmid DNA wasprepared using a QIAprep Spin Miniprep Kit (QIAGEN Inc., Valencia,Calif., USA). The inserted GH61 gene was confirmed by a DNA sequencingcompany (SinoGenoMax, Beijing). E. coli TOP10 strain, containingpGH61_(—)4950, was deposited with China General Microbiological CultureCollection Center (CGMCC) in Beijing, China on Feb. 23, 2011, andassigned accession number as CGMCC 4600.

Example 13 Characterization of the Genomic DNA Encoding GH61(3)Polypeptide

The genomic DNA sequence and deduced amino acid sequence of aThermomyces lanuginosus GH61 polypeptide coding sequence are shown inSEQ ID NO: 5 and SEQ ID NO: 6, respectively. The coding sequence is 881bp including the stop codon which is interrupted by 1 intron of 56 bp(nucleotides 335 to 390). The encoded predicted protein is 274 aminoacids. Using the SignalP program (Nielsen et al., 1997, supra), a signalpeptide of 22 residues was predicted. The predicted mature proteincontains 252 amino acids.

A comparative pairwise global alignment of amino acid sequences wasdetermined using the Needleman and Wunsch algorithm (Needleman andWunsch, 1970, supra) with gap open penalty of 10, gap extension penaltyof 0.5, and the EBLOSUM62 matrix. The alignment showed that the deducedamino acid sequence of the Thermomyces lanuginosus genomic DNA encodinga GH61 polypeptide shares 68.40% identity (excluding gaps) to a putativeendo-1,4-beta-glucanase from Aspergillus fumigatus (UNIPROT:B0XZE1_ASPFC).

Example 14 Expression of Thermomyces lanuginosus GH61(3) Gene inAspergillus oryzae

Aspergillus oryzae HowB101 (described in WO95/35385 example 1)protoplasts were prepared according to the method of Christensen et al.,1988, Bio/Technology 6: 1419-1422. Three μg of pGH61_(—)4950 were usedto transform Aspergillus oryzae HowB101.

The transformation of Aspergillus oryzae HowB101 with pGH61_(—)4950yielded about 20 transformants. Four transformants were isolated toindividual Minimal medium plates.

Four transformants were inoculated separately into 3 ml of YPM medium ina 24-well plate and incubated at 30° C., 150 rpm. After 3 daysincubation, 20 μl of supernatant from each culture were analyzed bySDS-PAGE using a NuPAGE® Novex 4-12% Bis-Tris Gel with2-(N-morpholino)ethanesulfonic acid (MES) (Invitrogen Corporation,Carlsbad, Calif., USA) according to the manufacturer's instructions. Theresulting gel was stained with INSTANT BLUE™ (Expedeon Ltd., BabrahamCambridge, UK). SDS-PAGE profiles of the cultures showed that themajority of the transformants had a band of approximate 45 kDa. Theexpression strain was designated as O5MMC.

A slant of one transformant was washed with 10 ml of YPM medium andinoculated into a 2 liter flask containing 400 ml of YPM medium togenerate broth for characterization of the enzyme. The culture washarvested on day 3 and filtered using a 0.45 μm DURAPORE Membrane(Millipore, Bedford, Mass., USA).

Example 15 Hydrolysis of Pretreated Corn Stover is Enhanced byThermomyces lanuginosus GH61(3) Polypeptide Having CellulolyticEnhancing Activity

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 0.048 g sulfuric acid/g drybiomass at 190° C. and 25% w/w dry solids for around 1 minute. Thewater-insoluble solids in the pretreated corn stover (PCS) contained 54%cellulose. Cellulose was determined by a two-stage sulfuric acidhydrolysis with subsequent analysis of sugars by high performance liquidchromatography (HPLC) using NREL Standard Analytical Procedure #002.Prior to enzymatic hydrolysis, the PCS was washed by water and groundusing a Multi Utility Grinder (Inno Concepts Inc., Roswell, Ga., USA)and sieved through a 450 μm screen by AS200 (Retsch, Haman, Germany).

The hydrolysis of pretreated corn stover was conducted using 1.8 ml,96-deep well plates (Beckman Instruments INC. Fullerton, USA) containinga total reaction mass of 1 g. The hydrolysis was performed with 7.8%total solids of washed pretreated corn stover, equivalent to 54 mg ofcellulose per ml, in 5 mM manganese sulfate—20 mM sodium acetate pH 5.0buffer containing a Trichoderma reesei base cellulase mixture(CELLUCLAST® supplemented with Aspergillus oryzae beta-glucosidase(recombinantly produced in Aspergillus oryzae according to WO 02/095014)available from Novozymes NS, Bagsvaerd, Denmark; the cellulasecomposition is designated herein in the Examples as “Trichoderma reeseicellulase composition”) at 2 mg per g of cellulose. Thermomyceslanuginosus GH61(3) polypeptide having cellulolytic enhancing activitywas separately added to the base cellulase mixture at ranging from 0 to90% of the concentration of base cellulase mixture. Plates were cappedusing a Capmat (Beckman Coulter, USA) and incubated at 50° C. and 60° C.for 118 hours with shaking at 150 rpm.

After 118 hours of incubation, 100 μl aliquots were removed and theextent of hydrolysis was assayed by high-performance liquidchromatography (HPLC) using the protocol described below.

For HPLC analysis, samples were filtered with a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. The sugar concentrationsof samples diluted in 5 mM H₂SO₄ were measured using a 4.6×250 mmAMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) by elution with 5 mM H₂SO₄ at a flow rate of 0.6 ml per minute at60° C. for 11 minutes, and quantification by integration of the glucoseand cellobiose signal from refractive index detection (CHEMSTATION®,AGILENT® 1200 HPLC, Agilent Technologies, Santa Clara, Calif., USA)calibrated by pure sugar samples. The resultant equivalents were used tocalculate the percentage of cellulose conversion for each reaction. Theextent of each hydrolysis was determined as the fraction of totalcellulose converted to cellobiose+glucose, and was not corrected forsoluble sugars present in pretreated corn stover liquor.

Measured sugar concentrations were adjusted for the appropriate dilutionfactor. Glucose and cellobiose were chromatographically separated andintegrated and their respective concentrations determined independently.However, to calculate total conversion the glucose and cellobiose valueswere combined. Fractional hydrolysis is reported as the overall massconversion to [glucose+cellobiose]/[total cellulose]. Triplicate datapoints were averaged.

The data demonstrated enhancement of hydrolysis by addition of theThermomyces lanuginosus GH61(3) polypeptide having cellulolyticenhancing activity. The addition of the GH61(3) polypeptide at 23%, 37%and 47% (w/w) enhanced hydrolysis by 24.0%, 25.8% and 26.7% glucanconversion in 118 hours of hydrolysis at 50° C., and enhanced hydrolysisby 82.7%, 13.3% and 0.0% glucan conversion in 118 hours of hydrolysis at60° C.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with China General Microbiological CultureCollection Center (CGMCC), NO. 1 West Beichen Road, Chaoyang District,Beijing 100101, China, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli (1) CGMCC 4601 Feb. 23,2011 E. coli (2) CGMCC 4602 Feb. 23, 2011 E. coli (3) CGMCC 4600 Feb.23, 2011

The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having cellulolytic enhancing activity,selected from the group consisting of:

(a) a polypeptide having at least 70% sequence identity to the maturepolypeptide of SEQ ID NO: 2, or at least 60% sequence identity to themature polypeptide of SEQ ID NO: 4, or at least 70% sequence identity tothe mature polypeptide of SEQ ID NO: 6;

(b) a polypeptide encoded by a polynucleotide that hybridizes under atleast high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) a full-lengthcomplementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 70%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof, or at least 60% sequence identity tothe mature polypeptide coding sequence of SEQ ID NO: 3 or the cDNAsequence thereof, or at least 70% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequencethereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4 orSEQ ID NO: 6 comprising a substitution, deletion, and/or insertion ofone or more (e.g., several) amino acids; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellulolytic enhancing activity.

[2] The polypeptide of paragraph 1, having at least 75% identity,preferably at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89% or at least 90% sequence identity to the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 6.

[3] The polypeptide of paragraph 2, having at least 91%, at least 92%,at least 93%, at least 94%, or at least 95% sequence identity to themature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6.

[4] The polypeptide of paragraph 3, having at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 6.

[5] The polypeptide of paragraph 1, having at least 65% identity, morepreferably at least 70% identity, most preferably at least 75% sequenceidentity to the mature polypeptide of SEQ ID NO: 4.

[6] The polypeptide of paragraph 5, having at least 80%, at least 81%,at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, at least 88%, at least 89% or at least 90% sequence identityto the mature polypeptide of SEQ ID NO: 4.

[7] The polypeptide of paragraph 6, having at least 91%, at least 92%,at least 93%, at least 94%, or at least 95% sequence identity to themature polypeptide of SEQ ID NO: 4.

[8] The polypeptide of paragraph 7, having at least 95% sequenceidentity to the mature polypeptide of SEQ ID NO: 4.

[9] The polypeptide of paragraph 8, having at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity to the maturepolypeptide of SEQ ID NO: 4.

[10] The polypeptide of paragraph 1, comprising or consisting of theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or afragment thereof having cellulolytic enhancing activity.

[11] The polypeptide of paragraph 10, comprising or consisting of theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

[12] The polypeptide of paragraph 10, comprising or consisting of themature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

[13] The polypeptide of paragraph 1, which is encoded by apolynucleotide that hybridizes under at least medium stringencyconditions, preferably at least medium-high, at least high, or at leastvery high stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) a full-lengthcomplementary strand of (i) or (ii).

[14] The polypeptide of paragraph 13, which is encoded by apolynucleotide that hybridizes under at least medium-high, at leasthigh, or at least very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:5, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) afull-length complementary strand of (i) or (ii).

[15] The polypeptide of paragraph 1, which is encoded by apolynucleotide having at least 70% sequence identity, preferably atleast 75% sequence identity, more preferably at least 80%, at least 81%,at least 82%, at least 83%, at least 84%, or at least 85% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 orSEQ ID NO: 5.

[16] The polypeptide of paragraph 15, which is encoded by apolynucleotide having at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, or at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1 or SEQ ID NO: 5.

[17] The polypeptide of paragraph 16, which is encoded by apolynucleotide having at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 1 or SEQ ID NO: 5.

[18] The polypeptide of paragraph 1, which is encoded by apolynucleotide having at least 65% sequence identity, more preferably atleast 70% sequence identity, most preferably at least 75% sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 3.

[19] The polypeptide of paragraph 18, which is encoded by apolynucleotide having at least 80%, at least 81%, at least 82%, at least83%, at least 84%, or at least 85% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 3.

[20] The polypeptide of paragraph 19, which is encoded by apolynucleotide having at least 86%, at least 87%, at least 88%, at least89%, or at least 90% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 3.

[21] The polypeptide of paragraph 20, which is encoded by apolynucleotide having at least 91%, at least 92%, at least 93%, at least94%, or at least 95% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 3.

[22] The polypeptide of paragraph 21, which is encoded by apolynucleotide having at least 96%, at least 97%, at least 98%, at least99%, or 100% sequence identity to the mature polypeptide coding sequenceof SEQ ID NO: 3.

[23] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising or consisting of the polynucleotide of SEQ IDNO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or a subsequence thereof encodinga fragment having cellulolytic enhancing activity.

[24] The polypeptide of paragraph 23, which is encoded by apolynucleotide comprising or consisting of the polynucleotide of SEQ IDNO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

[25] The polypeptide of paragraph 23, which is encoded by apolynucleotide comprising or consisting of the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.

[26] The polypeptide of paragraph 1, wherein the polypeptide is avariant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQID NO: 6 comprising a substitution, deletion, and/or insertion of one ormore (e.g., several) amino acids.

[27] The polypeptide of paragraph 1, which is encoded by thepolynucleotide contained in plasmid pGH61_(—)664 which is contained inE. coli CGMCC 4601, plasmid pGH61_(—)1590 which is contained in E. coliCGMCC 4602, or plasmid pGH61_(—)4950 which is contained in E. coli CGMCC4600.

[28] The polypeptide of any of paragraphs 1-27, wherein the maturepolypeptide is amino acids 23 to 272 of SEQ ID NO: 2, amino acids 22 to327 of SEQ ID NO: 4, or amino acids 23 to 274 of SEQ ID NO: 6.

[29] The polypeptide of any of paragraphs 1-28, wherein the maturepolypeptide coding sequence is nucleotides 67 to 869 of SEQ ID NO: 1,nucleotides 64 to 1036 of SEQ ID NO: 3, or nucleotides 67 to 878 of SEQID NO: 5 or the cDNA sequence thereof.

[30] An isolated polynucleotide encoding the polypeptide of any ofparagraphs 1-29.

[31] The isolated polynucleotide of paragraph 30, comprising at leastone mutation in the mature polypeptide coding sequence of SEQ ID NO: 1,SEQ ID NO: 3, or SEQ ID NO: 5, in which the mutant polynucleotideencodes the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ IDNO: 6, respectively.

[32] A nucleic acid construct comprising the polynucleotide of paragraph30 or 31 operably linked to one or more (e.g., several) controlsequences that direct the production of the polypeptide in an expressionhost.

[33] A recombinant expression vector comprising the nucleic acidconstruct of paragraph 32.

[34] A recombinant host cell comprising the nucleic acid construct ofparagraph 32.

[35] A method of producing the polypeptide of any of paragraphs 1-29,comprising:

(a) cultivating a cell, which in its wild-type form produces thepolypeptide, under conditions conducive for production of thepolypeptide; and

(b) recovering the polypeptide.

[36] A method of producing the polypeptide of any of paragraphs 1-29,comprising:

(a) cultivating a host cell comprising a nucleic acid constructcomprising a polynucleotide encoding the polypeptide under conditionsconducive for production of the polypeptide; and

(b) recovering the polypeptide.

[37] A method of producing a mutant of a parent cell, comprisingdisrupting or deleting a polynucleotide encoding the polypeptide, or aportion thereof, of any of paragraphs 1-29, which results in the mutantproducing less of the polypeptide than the parent cell.

[38] A mutant cell produced by the method of paragraph 37.

[39] The mutant cell of paragraph 38, further comprising a gene encodinga native or heterologous protein.

[40] A method of producing a protein, comprising:

(a) cultivating the mutant cell of paragraph 39 under conditionsconducive for production of the protein; and

(b) recovering the protein.

[41] The isolated polynucleotide of paragraph 30 or 31, obtained by (a)hybridizing a population of DNA under at least medium, at leastmedium-high, at least high, or at least very high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ IDNO: 3, or SEQ ID NO:5, (ii) the cDNA sequence contained in the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ IDNO:5, or (iii) a full-length complementary strand of (i) or (ii); and(b) isolating the hybridizing polynucleotide, which encodes apolypeptide having cellulolytic enhancing activity.

[42] The isolated polynucleotide of paragraph 41, obtained by (a)hybridizing a population of DNA under at least medium-high, at leasthigh, or at least very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO:5, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) afull-length complementary strand of (i) or (ii); and (b) isolating thehybridizing polynucleotide, which encodes a polypeptide havingcellulolytic enhancing activity.

[43] The isolated polynucleotide of paragraph 41 or 42, wherein themature polypeptide coding sequence is nucleotides 67 to 869 of SEQ IDNO: 1, nucleotides 64 to 1036 of SEQ ID NO: 3, or nucleotides 67 to 878of SEQ ID NO: 5.

[44] A method of producing a polynucleotide comprising a mutantpolynucleotide encoding a polypeptide having cellulolytic enhancingactivity, comprising:

(a) introducing at least one mutation into the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, wherein themutant polynucleotide encodes a polypeptide comprising or consisting ofthe mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;and

(b) recovering the polynucleotide comprising the mutant nucleotidesequence.

[45] A mutant polynucleotide produced by the method of paragraph 44.

[46] A method of producing a polypeptide, comprising:

(a) cultivating a cell comprising the mutant polynucleotide of paragraph45 encoding the polypeptide under conditions conducive for production ofthe polypeptide; and

(b) recovering the polypeptide.

[47] A method of producing the polypeptide of any of paragraphs 1-29,comprising:

(a) cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide; and

(b) recovering the polypeptide.

[48] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-29.

[49] A double-stranded RNA (dsRNA) molecule comprising a subsequence ofthe polynucleotide of paragraph 30 or 31, wherein optionally the dsRNAis an siRNA or an miRNA molecule.

[50] The double-stranded RNA (dsRNA) molecule of paragraph 49, which isabout 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplexnucleotides in length.

[51] A method of inhibiting the expression of a polypeptide havingcellulolytic enhancing activity in a cell, comprising administering tothe cell or expressing in the cell a double-stranded RNA (dsRNA)molecule, wherein the dsRNA comprises a subsequence of thepolynucleotide of paragraph 30 or 31.

[52] The method of paragraph 51, wherein the dsRNA is about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.

[53] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 22 of SEQ ID NO: 2, amino acids 1 to 21of SEQ ID NO: 4, or amino acids 1 to 22 of SEQ ID NO: 6.

[54] A nucleic acid construct comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 53, wherein the geneis foreign to the polynucleotide.

[55] A recombinant expression vector comprising the nucleic acidconstruct of paragraph 54.

[56] A recombinant host cell comprising the nucleic acid construct ofparagraph 54.

[57] A method of producing a protein, comprising: (a) cultivating therecombinant host cell of paragraph 56 under conditions conducive forproduction of the protein; and (b) recovering the protein.

[58] A composition comprising the polypeptide of any of paragraphs 1-29.

[59] The composition of paragraph 58, which is a cellulolytic enzymecomposition or a detergent composition.

[60] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of the polypeptide having cellulolytic enhancingactivity of any of paragraphs 1-29.

[61] The method of paragraph 60, wherein the cellulosic material ispretreated.

[62] The method of paragraph 60 or 61, wherein the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes or cellulasesselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[63] The method of any of paragraphs 60-62, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin; preferably the hemicellulase is one or more enzymes selectedfrom the group consisting of a xylanase, an acetylxylan esterase, aferuloyl esterase, an arabinofuranosidase, a xylosidase, and aglucuronidase.

[64] The method of any of paragraphs 60-63, further comprisingrecovering the degraded cellulosic material.

[65] The method of paragraph 64, wherein the degraded cellulosicmaterial is a sugar.

[66] The method of paragraph 65, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[67] A method for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the polypeptide having cellulolytic enhancing activity ofany of paragraphs 1-29; (b) fermenting the saccharified cellulosicmaterial with one or more (e.g., several) fermenting microorganisms toproduce the fermentation product; and (c) recovering the fermentationproduct from the fermentation.

[68] The method of paragraph 67, wherein the cellulosic material ispretreated.

[69] The method of paragraph 67 or 68, wherein the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes or cellulasesselected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[70] The method of any of paragraphs 67-69, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin; preferably the hemicellulase is one or more enzymes selectedfrom the group consisting of a xylanase, an acetylxylan esterase, aferuloyl esterase, an arabinofuranosidase, a xylosidase, and aglucuronidase.

[71] The method of any of paragraphs 67-70, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[72] The method of any of paragraphs 69-71, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[73] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more (e.g., several)fermenting microorganisms, wherein the cellulosic material issaccharified with an enzyme composition in the presence of a polypeptidehaving cellulolytic enhancing activity of any of paragraphs 1-29.

[74] The method of paragraph 73, wherein the fermenting of thecellulosic material produces a fermentation product.

[75] The method of paragraph 73 or 74, further comprising recovering thefermentation product from the fermentation.

[76] The method of any of paragraphs 73-75, wherein the cellulosicmaterial is pretreated before saccharification.

[77] The method of any of paragraphs 73-76, wherein the enzymecomposition comprises one or more (e.g., several) cellulolytic enzymesor cellulases selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[78] The method of any of paragraphs 73-77, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a polypeptide having cellulolytic enhancingactivity, a hemicellulase, an esterase, an expansin, a laccase, aligninolytic enzyme, a pectinase, a peroxidase, a protease, and aswollenin; preferably the hemicellulase is one or more enzymes selectedfrom the group consisting of a xylanase, an acetylxylan esterase, aferuloyl esterase, an arabinofuranosidase, a xylosidase, and aglucuronidase.

[79] The method of any of paragraphs 73-78, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[80] A whole broth formulation or cell culture composition comprisingthe polypeptide of any of paragraphs 1-13.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A method for producing a fermentation product,comprising: (a) saccharifying a cellulosic material with an enzymecomposition in the presence of a GH61 polypeptide having cellulolyticenhancing activity; (b) fermenting the saccharified cellulosic materialwith one or more fermenting microorganisms to produce the fermentationproduct; and (c) recovering the fermentation product from thefermentation; wherein the GH61 polypeptide having cellulolytic enhancingactivity is selected from the group consisting of: (i) a GH61polypeptide having at least 90% sequence identity to the maturepolypeptide of SEQ ID NO: 2, the mature polypeptide of SEQ ID NO: 4, orthe mature polypeptide of SEQ ID NO: 6; (ii) a GH61 polypeptide encodedby a polynucleotide that hybridizes under at least high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) the cDNA sequence contained inthe mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, orSEQ ID NO: 5, or (iii) a full-length complementary strand of (i) or(ii), wherein high stringency conditions are defined as prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 50% formamide, and washingthree times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.; (iii) aGH61 polypeptide encoded by a polynucleotide having at least 90%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof, the mature polypeptide codingsequence of SEQ ID NO: 3 or the cDNA sequence thereof, or the maturepolypeptide coding sequence of SEQ ID NO: 5 or the cDNA sequencethereof; and (iv) a fragment of the polypeptide of (i), (ii), or (iii),that has cellulolytic enhancing activity.
 2. The method of claim 1,wherein the cellulosic material is pretreated.
 3. The method of claim 1,wherein steps (a) and (b) are performed simultaneously in a simultaneoussaccharification and fermentation.
 4. The method of claim 1, wherein theenzyme composition comprises one or more enzymes selected from the groupconsisting of a cellulase, an AA9 polypeptide, a hemicellulase, a CIP,an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, apectinase, a protease, and a swollenin.
 5. The method of claim 4,wherein the cellulase is one or more enzymes selected from the groupconsisting of an endoglucanase, a cellobiohydrolase, and abeta-glucosidase.
 6. The method of claim 4, wherein the hemicellulase isone or more enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.
 7. The method of claim 1, wherein thefermentation product is an alcohol, an alkane, a cycloalkane, an alkene,an amino acid, a gas, isoprene, a ketone, an organic acid, orpolyketide.
 8. The method of claim 1, wherein the GH61 polypeptide hasat least 95% sequence identity to the mature polypeptide of SEQ ID NO:2, the mature polypeptide of SEQ ID NO: 4, or the mature polypeptide ofSEQ ID NO:
 6. 9. The method of claim 1, wherein the GH61 polypeptidecomprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQID NO: 6 or the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQID NO: 6; or a fragment thereof having cellulolytic enhancing activity.10. The method of claim 1, wherein the GH61 polypeptide is encoded by apolynucleotide having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof, the mature polypeptide coding sequence of SEQ ID NO: 3 or thecDNA sequence thereof, or the mature polypeptide coding sequence of SEQID NO: 5 or the cDNA sequence thereof.
 11. The method of claim 1,wherein the GH61 polypeptide is encoded by a polynucleotide comprisingthe mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNAsequence thereof, the mature polypeptide coding sequence of SEQ ID NO: 3or the cDNA sequence thereof, or the mature polypeptide coding sequenceof SEQ ID NO: 5 or the cDNA sequence thereof.
 12. A method of fermentinga cellulosic material, comprising: fermenting the cellulosic materialwith one or more fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellulolytic enhancing activity, wherein the GH61polypeptide having cellulolytic enhancing activity is selected from thegroup consisting of: (a) a GH61 polypeptide having at least 90% sequenceidentity to the mature polypeptide of SEQ ID NO: 2, the maturepolypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6;(b) a GH61 polypeptide encoded by a polynucleotide that hybridizes underat least high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, (ii) thecDNA sequence contained in the mature polypeptide coding sequence of SEQID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, or (iii) a full-lengthcomplementary strand of (i) or (ii), wherein high stringency conditionsare defined as prehybridization and hybridization at 42° C. in 5×SSPE,0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and50% formamide, and washing three times each for 15 minutes using 2×SSC,0.2% SDS at 65° C.; (c) a GH61 polypeptide encoded by a polynucleotidehaving at least 90% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof, the maturepolypeptide coding sequence of SEQ ID NO: 3 or the cDNA sequencethereof, or the mature polypeptide coding sequence of SEQ ID NO: 5 orthe cDNA sequence thereof; and (d) a fragment of the polypeptide of (a),(b), or (c), that has cellulolytic enhancing activity.
 13. The method ofclaim 12, wherein the cellulosic material is pretreated.
 14. The methodof claim 12, wherein the enzyme composition comprises one or moreenzymes selected from the group consisting of a cellulase, an AA9polypeptide, a hemicellulase, a CIP, an esterase, an expansin, aligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and aswollenin.
 15. The method of claim 14, wherein the cellulase is one ormore enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.
 16. The method of claim 14,wherein the hemicellulase is one or more enzymes selected from the groupconsisting of a xylanase, an acetylxylan esterase, a feruloyl esterase,an arabinofuranosidase, a xylosidase, and a glucuronidase.
 17. Themethod of claim 12, wherein the fermenting of the cellulosic materialproduces a fermentation product.
 18. The method of claim 17, furthercomprising recovering the fermentation product from the fermentation.19. The method of claim 18, wherein the fermentation product is analcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas,isoprene, a ketone, an organic acid, or polyketide.
 20. The method ofclaim 12, wherein the GH61 polypeptide has at least 95% sequenceidentity to the mature polypeptide of SEQ ID NO: 2, the maturepolypeptide of SEQ ID NO: 4, or the mature polypeptide of SEQ ID NO: 6.21. The method of claim 12, wherein the GH61 polypeptide comprises theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 orthe mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6;or a fragment thereof having cellulolytic enhancing activity.
 22. Themethod of claim 12, wherein the GH61 polypeptide is encoded by apolynucleotide having at least 95% sequence identity to the maturepolypeptide coding sequence of SEQ ID NO: 1 or the cDNA sequencethereof, the mature polypeptide coding sequence of SEQ ID NO: 3 or thecDNA sequence thereof, or the mature polypeptide coding sequence of SEQID NO: 5 or the cDNA sequence thereof.
 23. The method of claim 12,wherein the GH61 polypeptide is encoded by a polynucleotide comprisingthe mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNAsequence thereof, the mature polypeptide coding sequence of SEQ ID NO: 3or the cDNA sequence thereof, or the mature polypeptide coding sequenceof SEQ ID NO: 5 or the cDNA sequence thereof.